U.S. patent application number 16/873603 was filed with the patent office on 2021-11-25 for methods and compositions for enhancing synthesis secretion and transport of collagen to increase wound strength.
This patent application is currently assigned to North Cell Pharmaceuticals, Inc.. The applicant listed for this patent is Alain Martin. Invention is credited to Alain Martin.
Application Number | 20210361604 16/873603 |
Document ID | / |
Family ID | 1000005032735 |
Filed Date | 2021-11-25 |
United States Patent
Application |
20210361604 |
Kind Code |
A1 |
Martin; Alain |
November 25, 2021 |
Methods and Compositions For Enhancing Synthesis Secretion and
Transport of Collagen to Increase Wound Strength
Abstract
A method for, and medicinal composition for, treatment of
mammalian cells for enhancing synthesis, secretion and transport of
collagen to increase wound strength, that includes magnesium
pyruvate; calcium pyruvate; L proline; citrulline: and a liquid
carrier.
Inventors: |
Martin; Alain; (Flemington,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Martin; Alain |
Flemington |
NJ |
US |
|
|
Assignee: |
North Cell Pharmaceuticals,
Inc.
|
Family ID: |
1000005032735 |
Appl. No.: |
16/873603 |
Filed: |
May 19, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/198 20130101;
A61K 45/06 20130101; A61P 17/02 20180101; A61K 9/08 20130101; A61K
31/401 20130101 |
International
Class: |
A61K 31/198 20060101
A61K031/198; A61K 31/401 20060101 A61K031/401; A61K 9/08 20060101
A61K009/08; A61P 17/02 20060101 A61P017/02 |
Claims
1. A medicinal composition for treatment of mammalian cells,
comprising: a) magnesium pyruvate; b) calcium pyruvate; c) L
proline; d) and citrulline: and, e) a liquid carrier.
2. The medicinal composition of claim 1 wherein said magnesium
pyruvate and said calcium pyruvate are each in the amount of about
0.1% to about 5.0%, based on total weight of the composition.
3. The medicinal composition of claim 2 wherein said magnesium
pyruvate and said calcium pyruvate are each in the amount of about
0.2% to about 4.0%, based on total weight of the composition.
4. The medicinal composition of claim 3 wherein said magnesium
pyruvate and said calcium pyruvate are each in the amount of about
0.5% to about 4.0%, based on total weight of the composition.
5. The medicinal composition of claim 1 wherein said L proline and
said citrulline are each in the amount of about 0.1% to about 3.0%,
based on total weight of the composition.
6. The medicinal composition of claim 5 wherein said L proline and
said citrulline are each in the amount of about 0.2% to about 2.5%,
based on total weight of the composition.
7. The medicinal composition of claim 1 wherein said citrulline is
selected from the group consisting of L citrulline, citrulline
malate and combinations thereof
8. The medicinal composition of claim 1 wherein said composition
has a carrier selected from the group consisting of water, oil,
water-based carriers, oil-based carriers, gels, emulsions, lotions,
creams, solutions and petrolatum.
9. The medicinal composition of claim 1 wherein said at least one
additional active constituent is selected from the group consisting
of antioxidants, antibacterial agents, alpha keto acids, antiviral
agents, antifungal agents, antihistamine agents, cancer drugs, HIV
drugs non-steroidal anti-inflammatory agents, antioxidant agents,
fatty acids, steroids, and mixtures thereof.
10. The medicinal composition of claim 1 wherein said alpha-keto
acids have four or more carbon atoms and are selected from the
group consisting of oxaloacetate, alpha-keto-glutarate,
alpha-keto-isovalerate, alpha keto-butyric acid, alpha keto-adipic
acid, alpha.-keto-caproic acid, salts thereof, and mixtures
thereof.
11. A treatment method for treatment of mammalian cells for
enhancing the synthesis of collagen, adhesion molecules, and
elastin to increase wound strength, wound texture, pigmentation and
nitric oxide, wherein the wounds and diseases are selected from the
group consisting patients with wrinkles, aging skin, cellulitis,
wounds and diseases which are selected from the group consisting of
infected and noninfected wounds, bacterial infections, fungal
infections, viral infections, cold sores, thermal burns, sunburns,
chemical burns, surgical wounds, psoriasis, eczema, decubitus
ulcers, cancer, diabetic ulcers, dermatoses, inflammatory diseases,
and wounds resulting from laser treatment, micro-wounded skins,
scratchs, micro-needling, abrasions, laceration, puncture, dry
skin, contact with irritants, contact with allergens or other
substances that are harmful to skin or skin cells, which comprises:
applying a therapeutically effective amount of a medicinal
composition to mammalian cells, said medicinal composition
including: a) magnesium pyruvate; b) calcium pyruvate; c) L
proline; d) citrulline: and, e) a liquid carrier.
12. The method of claim 11 wherein said magnesium pyruvate and said
calcium pyruvate are each in the amount of about 0.1% to about
5.0%, based on total weight of the composition.
13. The method of claim 12 wherein said magnesium pyruvate and said
calcium pyruvate are each in the amount of about 0.2% to about
4.0%, based on total weight of the composition.
14. The method of claim 13 wherein said magnesium pyruvate and said
calcium pyruvate are each in the amount of about 0.5% to about
4.0%, based on total weight of the composition.
15. The method of claim 11 wherein said L proline and said
citrulline are each in the amount of about 0.1% to about 3.0%,
based on total weight of the composition.
16. The method of claim 15 wherein said L proline and said
citrulline are each in the amount of about 0.2% to about 2.5%,
based on total weight of the composition.
17. The method of claim 11 wherein said citrulline is selected from
the group consisting of L citrulline, citrulline malate and
combinations thereof
18. The method of claim 11 wherein said composition has a carrier
selected from the group consisting of water, oil, water-based
carriers, oil-based carriers, gels, emulsions, lotions, creams,
solutions and petrolatum.
19. The method of claim 11 wherein said at least one additional
active constituent is selected from the group consisting of
antioxidants, antibacterial agents, alpha keto acids, antiviral
agents, antifungal agents, antihistamine agents, cancer drugs, HIV
drugs non-steroidal anti-inflammatory agents, antioxidant agents,
fatty acids, steroids, and mixtures thereof.
20. The method of claim 19 wherein said alpha-keto acids have four
or more carbon atoms and are selected from the group consisting of
oxaloacetate, alpha-keto-glutarate, alpha-keto-isovalerate, alpha
keto-butyric acid, alpha keto-adipic acid, alpha-keto-caproic acid,
salts thereof, and mixtures thereof.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] The present application has no related pending patent
applications.
BACKGROUND OF INVENTION
Field of Invention
[0002] The present invention relates to a method and composition
for enhancing the synthesis, secretion and transport of collagen,
adhesion molecules and elastin to wound sites needed to increase
wound strength, wound texture and pigmentation. This invention has
demonstrated that a certain combination of salts of pyruvate with L
proline and citrulline were synergistic and enhanced the synthesis,
secretion and transport of collagen, adhesion molecules, and
elastin to wound sites that increased wound strength, wound texture
and pigmentation.
Description of Related Art
[0003] The following outlines the present invention and the history
of various prior art treatments and patents are representative of
the field pertaining to the present invention:
[0004] The present invention has demonstrated that a certain
combination of salts of pyruvate with L proline and citrulline were
synergistic and enhanced the synthesis, secretion and transport of
collagen, adhesion molecules, and elastin to wound sites that
increased wound strength, wound texture and pigmentation. The
combination of calcium pyruvate and magnesium pyruvate with the
addition of L proline and L-citrulline enhanced membrane synthesis,
secretion and transport type 1 collagen, cellular adhesion
molecules, and elastin at wound sites that increase wound strength,
wound texture and pigmentation. The magnesium and calcium salts of
pyruvate, with L proline and L citrulline (SRF--Skin resuscitation
Factor) was superior over standard and current sodium pyruvate
wound healing products and formula. The use of this formula (SRF)
clearly demonstrated that it can be used to produce better efficacy
in patients with wrinkles, aging skin, cellulitis, wounds and
diseases which are selected from the group consisting of infected
and noninfected wounds, bacterial infections, fungal infections,
cellulite, viral infections, cold sores, thermal burns, sunburns,
chemical burns, surgical wounds, psoriasis, eczema, decubitus
ulcers, cancer, diabetic ulcers, dermatoses, inflammatory diseases,
and wounds resulting from laser treatment, micro-wounded skins,
scratchs, micro-needling, abrasions, laceration, puncture, dry
skin, contact with irritants, contact with allergens or other
substances that are harmful to skin or skin cells.
[0005] Membrane transport systems are the key to the synthesis,
secretion and transport of key molecules to maintain health for all
organs and to enhance wound repair and increase wound strength,
texture and pigmentation. Wounds are internal or external bodily
injuries or lesions caused by mechanical, chemical, viral,
bacterial or thermal means, which disrupt the normal continuity of
structures. Such bodily injuries include contusions, which are
wounds in which the skin is unbroken; incisions, i.e., which are
wounds in which the skin is broken by a cutting instrument; and
lacerations, which are wounds in which the skin is broken by a dull
or blunt instrument. Membrane transport systems are the key to the
synthesis and secretion of key molecules to maintain health for all
organs and to enhance wound repair and wound strength, texture and
pigmentation. Most wounds also produce pain, swelling, itching,
ischemia, crusting, erythema, scarring and all wounds loose wound
strength, texture and pigmentation. The rate of healing,
(reepithialization) does not necessarily correlate with wound
strength, texture or pigmentation. You can have a wound that heals
quickly, but has low wound strength allowing the wound to reopen
again and again.
[0006] Wound strength: Cell Adhesion Molecules (CAMs) are proteins
located on the cell surface involved with the binding with other
cells or with the extracellular matrix (ECM) in the process called
cell adhesion that enhance healing and wound strength. Essentially,
cell adhesion molecules help cells stick to each other and to their
surroundings. These proteins are typically transmembrane receptors
and are composed of three domains: an intracellular domain that
interacts with the cytoskeleton, a transmembrane domain, and an
extracellular domain that interacts either with other CAMs of the
same kind (homophilic binding) or with other CAMs or the
extracellular matrix (heterophilic binding).
[0007] Most of the CAMs belong to four protein families: Ig
(immunoglobulin) superfamily (IgSF CAMs), the integrins, the
cadherins, and the selectins. One classification system involves
the distinction between calcium-independent CAMs and
calcium-dependent CAMs. The Integrins are a family of heterophilic
CAMs that bind IgSF CAMs or the extracellular matrix. They are
heterodimers, called alpha and beta. Eighteen different alpha
subunits that combine with 8 different beta subunits to form
twenty-four known integrins; however not all combinations are
observed. The cadherin's are a family of homophylic CAMs,
Ca.sup.2+-dependent.
[0008] Melanocytes are melanin-producing cells located in the
bottom layer of the skin's epidermis, the middle layer of the eye
(the uvea), the inner ear, bones, and heart. Melanin is the pigment
primarily responsible for skin color. Once synthesized, melanin is
contained in a special organelle called a melanosome and moved
along arm-like structures called dendrites, so as to reach the
keratinocytes. Through a process called melanogenesis, these cells
produce melanin, which is a pigment found in the skin, eyes, and
hair. This melanogenesis leads to a long-lasting pigmentation,
which is in contrast to the pigmentation that originates from
oxidation of already-existing melanin.
[0009] One of fibroblasts' most important duties is the production
of collagen. Collagen deposition is important because it increases
the strength of the wound; before it is laid down, the only thing
holding the wound closed is the fibrin-fibronectin clot, which does
not provide much resistance to traumatic injury. Also, cells
involved in inflammation, angiogenesis, and connective tissue
construction attach to, grow and differentiate on the collagen
matrix laid down by fibroblasts. Type III collagen and fibronectin
are generally beginning to be produced in appreciable amounts at
somewhere between approximately 10 hours and 3 days, depending
mainly on wound size. Their deposition peaks at one to three weeks.
They are the predominating tensile substances until the later phase
of maturation, in which they are replaced by the stronger type I
collagen. Even as fibroblasts are producing new collagen,
collagenases and other factors degrade it. Shortly after wounding,
synthesis exceeds degradation so collagen levels in the wound rise,
but later production and degradation become equal so there is no
net collagen gain. This homeostasis signals the onset of the later
maturation phase. Granulation gradually ceases and fibroblasts
decrease in number in the wound once their work is done. At the end
of the granulation phase, fibroblasts begin to commit apoptosis,
converting granulation tissue from an environment rich in cells to
one that consists mainly of collagen. Mitochondria have a direct
impact on the synthesis of cellular adhesion molecules and
collagen. Increasing the synthesis of collagen is also a critical
aspect of decreasing wrinkles.
[0010] Elastin, which is not collagen, is a highly elastic protein
in connective tissue and allows many tissues in the body to resume
their shape after stretching or contracting. Elastin helps skin to
return to its original position when it is poked or pinched.
Elastic fiber is composed mainly of an amorphous component, which
is extensively cross-linked elastin, and a fibrillar component,
which are primarily the microfibrils such as fibrillin, both of
which are made of simple amino acids such as glycine, valine,
alanine, and proline.
[0011] Eczema or atopic dermatitis is a form of dermatitis, or
inflammation of the epidermis (the outer layer of the skin). These
types of diseases all suffer from weak wound strength. The term
eczema is broadly applied to a range of persistent skin conditions.
These include dryness and recurring skin rashes that are
characterized by one or more of these symptoms: redness, skin edema
(swelling), itching and dryness, crusting, flaking, blistering,
cracking, oozing, or bleeding. Areas of temporary skin
discoloration may appear and are sometimes due to healed injuries.
Scratching open a healing lesion may result in scarring and may
enlarge the rash.
[0012] The type of eczema may be described by location (e.g., hand
eczema), by specific appearance (eczema craquele or discoid), or by
possible cause (varicose eczema). Further adding to the confusion,
many sources use the term eczema for the most common type of eczema
(atopic dermatitis) interchangeably. The classifications below is
ordered by incidence frequency. Atopic eczema, Contact dermatitis,
Xerotic eczema, Seborrheic dermatitis, Dyshidrosis, Discoid eczema,
Venous eczema, Dermatitis herpetiformis, Neurodermitis,
Autoeczematization. Eczema can be exacerbated by dryness of the
skin. Moisturizing is one of the most important self-care
treatments for eczema.
[0013] Decubitus ulcers, Pressure ulcers, also known as pressure
sores, bedsores and decubitus ulcers, are localized injuries to the
skin and/or underlying tissue that usually occur over a bony
prominence as a result of pressure, or pressure in combination with
shear and/or friction. The most common sites are the skin overlying
the sacrum, coccyx, heels or the hips, but other sites such as the
elbows, knees, ankles or the back of the cranium can be
affected.
[0014] Pressure ulcers occur due to pressure applied to soft tissue
resulting in completely or partially obstructed blood flow to the
soft tissue. Shear is also a cause, as it can pull on blood vessels
that feed the skin. Pressure ulcers most commonly develop in
individuals who are not moving about, such as being bedridden or
are confined to a wheelchair. It is widely believed that other
factors can influence the tolerance of skin for pressure and shear,
thereby increasing the risk of pressure ulcer development. These
factors are protein-calorie malnutrition, microclimate (skin
wetness caused by sweating or incontinence), diseases that reduce
blood flow to the skin, such as arteriosclerosis, or diseases that
reduce the sensation in the skin, such as paralysis or neuropathy.
The healing of pressure ulcers may be slowed by the age of the
person, medical conditions (such as arteriosclerosis, diabetes or
infection), smoking or medications such as anti-inflammatory
drugs.
[0015] External pressure applied over an area of the body,
especially over the bony prominences can result in obstruction of
the blood capillaries, which deprives tissues of oxygen and
nutrients, causing ischemia (deficiency of blood in a particular
area), hypoxia (inadequate amount of oxygen available to the
cells), edema, inflammation, and, finally, necrosis and ulcer
formation. In all pressure ulcers, especially in decubitus ulcers,
cellular adhesion molecules are produced at low levels and wound
strength never occurs due to the inhibition and damage done to
mitochondria and the inability of mitochondria to synthesize
membrane phospholipids needed to enhance the synthesis of cellular
adhesion molecules, collagen deposition and wound strength.
[0016] Aging Skin. Aging of skin is an intricate biological process
consisting of two types. While intrinsic or chronological aging is
an inevitable process, photoaging involves the premature aging of
skin occurring due to cumulative exposure to ultraviolet radiation.
Chronological and photoaging both have clinically differentiable
manifestations. Skin aging is influenced by several factors
including genetics, environmental exposure (UV radiation,
xenobiotics, and mechanical stress), hormonal changes and metabolic
processes (generation of reactive chemical compounds such as
activated oxygen species, sugars and aldehydes). All factors
together act on the alterations of skin structure, function, and
appearance. Yet solar UV radiation unquestionably is the single
major factor responsible for skin aging. Clinically, the
intrinsically aged skin is atrophic, which may result in prominence
of vasculature and loss of elasticity. The stratum corneum remains
relatively unchanged but the epidermis thins with a flattening of
the dermo-epidermal junction expressing an increased fragility of
the skin. There is considerable decrease in dermal thickness and
vascularity as well as a reduction in the number and biosynthetic
capacity of the fibroblast resulting in delayed wound healing, loss
of wound strength and texture. With an increasing age, there is a
progressive decline in the response of keratinocytes and
fibroblasts to growth factors, decreasing the proliferative
capacity.
[0017] Photoaging is the superimposition of photodamage on
intrinsically aged skin generally bringing about premature aging.
This specific damage occurs by chronic (multiple) exposure of the
skin to UV light. Clinically, the skin becomes coarse; epidermis
thickens (hyperplasia) initially and then thins (atrophy), there is
laxity, sallowness with wrinkles, irregular hyperpigmentation,
lentigines, and telangiectasias, acrochordon, and ruby spots),
"premalignant" lesions (actinic keratosis, lentigo maligna), and
malignant lesions (basal and squamous cell carcinomas and malignant
melanomas) on chronically exposed skin found in the face, hands and
neck regions. Blood vessels become dilated and twisted
(telangiectasia) and finally very sparse, while their walls are
initially thickened and later thinned. UV irradiation of the skin
increases the reactive oxygen species and decreases the endogenous
antioxidant enzymes. This increased production of ROS alters gene
and protein structure and function leading to skin damage.
[0018] Various natural and synthetic retinoids have been explored
for the treatment of aging and many of them have shown histological
and clinical improvement, but most of the studies have been carried
out in patients presenting with photoaged skin. Retinol family
comprises vitamin A (retinol) and its natural derivatives such as
retinaldehyde, retinoic acid, and retinyl esters, as well as a
large number of synthetic derivatives. Retinoids are required for a
vast number of biological processes. In particular, they are
involved in embryogenesis, reproduction, vision, growth,
inflammation, differentiation, proliferation, and apoptosis.
Retinal is an essential part of the rhodopsin pigment, necessary
for visions. Retinoids are found in the keratinocytes in two forms:
retinol and retinyl esters--probably the storage form. Although
retinoids show promise in the treatment of skin aging, irritant
reactions such as burning, redness, dryness, swelling, crusting,
blistering, scaling or dermatitis associated with retinoid therapy
limit their acceptance by patients.
[0019] U.S. Pat. No. 5,296,370, issued to Martin, et al., discloses
therapeutic compositions for preventing and reducing injury to
mammalian cells and increasing the resuscitation rate of injured
mammalian cells. In one embodiment, the therapeutic composition
comprises (a) a pyruvate, (b) an antioxidant, and (c) a mixture of
saturated and unsaturated fatty acids.
[0020] U.S. Pat. No. 5,863,938, issued to Martin, discloses a
therapeutic antibacterial wound-healing composition comprising an
effective amount of an antibacterial agent and a wound-healing
composition consisting of (a) pyruvate (b) an antioxidant, and (c)
a mixture of fatty acids. This patent teaches the use of pyruvate,
vitamin E and fatty acids to treat wounds.
[0021] U.S. Pat. No. 7,122,578 (Martin) discloses a method for
treating wounds, injuries, diseases and dermatological disease
states in mammals caused by mammalian cells involved in the
inflammatory response comprising contacting the mammalian cells
with an antioxidant reactive oxygen species mediator selected from
the group consisting of .alpha.-keto acids used singly or in
combination in an amount capable of reducing the undesired
inflammatory conditions. The only combination of alpha keto acids
shown to be synergistic was pyruvate with alpha keto isovalerate
with fatty acids.
[0022] U.S. Pat. No. 8,076,373 (Martin) teaches the use of alpha
keto acids to protect drugs such as antivirals, antibacterials,
antifungals, and anticancer drugs that are attacked by oxygen
radicals especially peroxynitrite. In this patent the use of
pyruvate with other keto acids without vitamin E and fatty acids
was shown to protect drugs form peroxynitrite.
[0023] The patented therapeutic compositions listed above report
the use alpha keto acids as antioxidants that neutralize the
negative effects of reactive oxygen radicals (sodium pyruvate,
alpha ketoglutarate, keto isovalerate and vitamin E) at wound
sites, with the addition of fatty acids for membrane repair, which
enhance the rate of wound healing.
[0024] Enhancing the rate of wound healing does not guarantee an
increase in wound strength, texture, elasticity and pigmentation at
wound sites. None of the compositions or inventions to date provide
a method for enhancing the synthesis of key adhesion molecules,
skin phospholipids, elastin and type 1 collagen, that increases
wound strength, texture and pigmentation. By using the synergistic
combination of calcium pyruvate, magnesium pyruvate, in combination
with the correct concentrations and ratios of L proline and
L-citrulline (SRF) the transport and synthesis of adhesion
molecules, phospholipids, elastin and type 1 collagen, increased
wound strength, wound texture and pigmentation, while decreasing
wound scars. Also, SRF increased the synthesis of Collagen to
eliminate wrinkles, increased wound strength, wound texture and
pigmentation.
[0025] The disclosures referred to herein to illustrate the
background of the invention and to provide additional detail with
respect to its practice are incorporated herein by reference and,
for convenience, are referenced in the following text and
respectively grouped in the appended bibliography.
SUMMARY OF THE INVENTION
[0026] The present invention is directed to a medicinal composition
for treatment of mammalian cells, comprising: magnesium pyruvate;
calcium pyruvate; L proline; citrulline: and, a liquid carrier. In
some embodiments of the present invention medicinal composition,
the magnesium pyruvate and the calcium pyruvate are each in the
amount of about 0.1% to about 5.0%, based on total weight of the
composition. In some preferred embodiments, the magnesium pyruvate
and the calcium pyruvate are each in the amount of about 0.2% to
about 4.0%, based on total weight of the composition. In some
embodiments, magnesium pyruvate and the calcium pyruvate are each
in the amount of about 0.5% to about 4.0%, based on total weight of
the composition. In some embodiments of the present invention
medicinal composition, the L proline and the citrulline are each in
the amount of about 0.1% to about 3.0%, based on total weight of
the composition. In some embodiments, the L proline and the
citrulline are each in the amount of about 0.2% to about 2.5%,
based on total weight of the composition. In some embodiments, the
citrulline is selected from the group consisting of L citrulline,
citrulline malate and combinations thereof. In some embodiments,
the composition has a carrier selected from the group consisting of
water, oil, water-based carriers, oil-based carriers, gels,
emulsions, lotions, creams, solutions and petrolatum. In some
embodiments, there is at least one additional active constituent
that is selected from the group consisting of antioxidants,
antibacterial agents, alpha keto acids, antiviral agents,
antifungal agents, antihistamine agents, cancer drugs, HIV drugs
non-steroidal anti-inflammatory agents, antioxidant agents, fatty
acids, steroids, and mixtures thereof. In some embodiments, the
alpha-keto acids have four or more carbon atoms and are selected
from the group consisting of oxaloacetate, alpha-keto-glutarate,
alpha-keto-isovalerate, alpha keto-butyric acid, alpha keto-adipic
acid, alpha-keto-caproic acid, salts thereof, and mixtures thereof.
In other embodiments, the present invention is a method utilizing
the components described herein in this paragraph. Thus, the
present invention treatment method for treatment of mammalian cells
for enhancing the synthesis of collagen, adhesion molecules, and
elastin to increase wound strength, wound texture, pigmentation and
nitric oxide, wherein the wounds and diseases are selected from the
group consisting patients with wrinkles, aging skin, cellulitis,
wounds and diseases which are selected from the group consisting of
infected and noninfected wounds, bacterial infections, fungal
infections, viral infections, cold sores, thermal burns, sunburns,
chemical burns, surgical wounds, psoriasis, eczema, decubitus
ulcers, cancer, diabetic ulcers, dermatoses, inflammatory diseases,
and wounds resulting from laser treatment, micro-wounded skins,
scratchs, micro-needling, abrasions, laceration, puncture, dry
skin, contact with irritants, contact with allergens or other
substances that are harmful to skin or skin cells, involves
applying a therapeutically effective amount of a medicinal
composition to mammalian cells, the medicinal composition
including: a) magnesium pyruvate; b) calcium pyruvate; c) L
proline; d) citrulline: and, e) a liquid carrier. The various
embodiments of the constituents are described above in this
paragraph.
BRIEF DESCRIPTION OF THE DRAWING
[0027] FIG. 1 shows a chart of a present invention wound healing
study, % re-epithelization in 25 subjects.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention provides a method for enhancing the
synthesis, secretion and transport of key components including
adhesion molecules, elastin and type 1 collagen to increase wound
strength, texture and pigmentation, by forcing fibroblasts to
synthetize the correct type of collagen that will reduce scar
formations and to stimulate melanocytes to synthesize melanin. The
composition method also enhances the production of phospholipids
needed to protect mitochondria and cells. The method comprises
contacting the mammalian cells with a therapeutically effective
amount of the synergistic mixture, wherein the synergistic mixture
is selected from the group consisting of certain salts of
pyruvates, magnesium and calcium, L proline and L-citrulline (this
formula is called Skin Resuscitation Factor (SRF)) wherein the
treatment results in the up regulation, transport and synthesis of
key components of cellular membranes that enhance wound strength,
wound texture and pigmentation at wound sites. Magnesium and
calcium, are an essential element in biological systems. Magnesium
occurs typically as the Mg.sup.2+ ion. It is an essential mineral
nutrient (i.e., element) for life and is present in every cell type
in every organism. For example, ATP (adenosine triphosphate), the
main source of energy in cells, must be bound to a magnesium ion in
order to be biologically active. What is called ATP is often
actually Mg-ATP. As such, magnesium plays a role in the stability
of all polyphosphate compounds in the cells, including those
associated with the synthesis of DNA and RNA. Over 300 enzymes
require the presence of magnesium ions for their catalytic action,
including all enzymes utilizing or synthesizing ATP, or those that
use other nucleotides to synthesize DNA and RNA. Thus, the calcium
and magnesium pyruvate are essential to this process, not sodium
pyruvate or any of the other salts of pyruvate to enhance the
synthesis, secretion and transport of key components including
adhesion molecules, elastin and type 1 collagen to increase wound
strength, texture and pigmentation, and healing. The rate of
healing, (reepithelization) does not necessarily correlate with
wound strength, texture or pigmentation. You can have a wound that
heals quickly, but has low wound strength allowing the wound to
reopen again and again.
[0029] Previous data from previous experiments demonstrated that
alpha-keto acids can mitigate the damage produced from the toxic
drug by-products caused by oxygen radicals including peroxynitrite,
and can decrease the inflammatory process, speed healing and
increase the efficacy of the drug at the wound site. Unexpectedly,
the combination of calcium pyruvate and magnesium pyruvate with L
proline and L-citrulline (SRF) were synergistic in enhancing
membrane transport systems to increase the synthesis, secretion and
transport of cellular molecules including adhesion molecules, skin
phospholipids, elastin and type 1 collagen that enhanced
re-epithelization, wound strength, wound texture and pigmentation.
All combinations of sodium pyruvate with alpha keto-butyric acid,
alpha keto-adipic acid, alpha.-keto-caproic acid, alpha keto
isovalerate, alpha ketoglutarate, and mixtures thereof, were not as
effective as SRF in enhancing the synthesis, secretion and
transport of adhesion molecules, collagen and elastin needed for
enhanced wound strength, wound texture and pigmentation. SRF
eliminated all the irritation and enhanced re-epithelization and
wound strength of skin by over 40% over sodium pyruvate alone or in
combination with other alpha keto acids and fatty acids, when
formulated into skin lotions to treat various wounds including
dermabrasion, cuts, scratches, lacerations, punctures, contusions,
post laser wounds, surgery wounds, UV damage, sunburns, dry weather
wounds, bacterial infection wounds, fungus infection wounds, virus
infection wounds, irritant wounds, allergen wounds, and wounds from
substances harmful micro wounds from dry skin. Even though other
salts of pyruvic acid like calcium or potassium etc have been
suggested, no one to date has evaluated the different salts
individually or in combination for their ability to enhance wound
healing. To evaluate the irritation or toxicity levels of these
salts a formulation of each salt of pyruvate was tested against the
standard formulations with sodium pyruvate that was shown to be non
toxic in humans and animal studies. Potassium pyruvate, and calcium
pyruvate, produced similar wound healing results in patients as the
sodium pyruvate formulations. The zinc pyruvate, magnesium
pyruvate, manganese pyruvate, aluminum pyruvate, ammonium pyruvate,
and lithium pyruvate did produce slight increases in healing but
did not achieve the results that sodium pyruvate produced, and they
were irritating and possibly toxic. The combination of calcium
pyruvate, magnesium pyruvate and L proline and citrulline was
superior to all other combinations.
[0030] As used herein, the following terms have the given
meanings:
[0031] The term "cytotoxicity", as used herein, means a condition
caused by a cytotoxic agent that injures the cell. Injured cells do
not readily proliferate because injured cells expend all energy on
cellular repair. Aiding cellular repair promotes cellular
proliferation.
[0032] The term "injured cell", as used herein, means a cell that
has any cellular activity disrupted for any reason. For example, an
injured cell may be a cell that has injured membranes or damaged
DNA, RNA, and/or ribosomes. For example, a cell which has (a)
injured membranes so that transport through the membranes is
diminished resulting in an increase in toxins and normal cellular
wastes inside the cell and a decrease in nutrients and other
components necessary for cellular repair inside the cell (b) an
increase in concentration of oxygen radicals inside the cell
because of the decreased ability of the cell to produce
antioxidants and enzymes, or (c) damaged DNA, RNA, and ribosomes
which must be repaired or replaced before normal cellular functions
can be resumed.
[0033] The term "metabolite, as used herein, refers to any
substance produced by metabolism or by a metabolic process.
"Metabolism," as used herein, refers to the various chemical
reactions involved in the transformation of molecules or chemical
compounds occurring in tissue and the cells therein.
[0034] The term "pharmaceutically acceptable", as used herein, such
as pharmaceutically acceptable carrier, excipient, etc. means
pharmacologically acceptable and substantially non-toxic to the
subject to which the particular compound is administered.
[0035] The term "pharmaceutically acceptable salt", as used herein,
refers to conventional acid-addition salts or base-addition salts
that retain the biological effectiveness and properties of the
compounds of the present invention and are formed from suitable
non-toxic organic or inorganic acids or organic or inorganic bases.
Sample acid-addition salts include those derived from inorganic
acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid,
sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and
those derived from organic acids such as p-toluenesulfonic acid,
salicylic acid, methanesulfonic acid, oxalic acid, succinic acid,
citric acid, malic acid, lactic acid, fumaric acid, and the like.
Sample base-addition salts include those derived from ammonium,
potassium, sodium and, quaternary ammonium hydroxides, such as for
example, tetramethylammonium hydroxide. Chemical modification of a
pharmaceutical compound (i.e. drug) into a salt is a technique well
known to pharmaceutical chemists to obtain improved physical and
chemical stability, hydroscopicity, flowability and solubility of
compounds. See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms
and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and
1456-1457.
[0036] The term "prodrug", as used herein, refers to compounds,
which undergo biotransformation prior to exhibiting their
pharmacological effects. The chemical modification of drugs to
overcome pharmaceutical problems has also been termed "drug
latentiation." Drug latentiation is the chemical modification of a
biologically active compound to form a new compound, which upon in
vivo enzymatic attack will liberate the parent compound. The
chemical alterations of the parent compound are such that the
change in physicochemical properties will affect the absorption,
distribution and enzymatic metabolism. The definition of drug
latentiation has also been extended to include nonenzymatic
regeneration of the parent compound. Regeneration takes place as a
consequence of hydrolytic, dissociative, and other reactions not
necessarily enzyme mediated. The terms prodrugs, latentiated drugs,
and bioreversible derivatives are used interchangeably. By
inference, latentiation implies a time lag element or time
component involved in regenerating the bioactive parent molecule in
vivo. The term prodrug is general in that it includes latentiated
drug derivatives as well as those substances, which are converted
after administration to the actual substance, which combines with
receptors. The term prodrug is a generic term for agents, which
undergo biotransformation prior to exhibiting their pharmacological
actions.
[0037] The term "reactive oxygen species", as used herein, means
activated oxygen species such as superoxide (O.sub.2.sup.-),
hydrogen peroxide (H.sub.2O.sub.2), hydroxyl radicals (OH), and
singlet oxygen (.sup.1O.sub.2). Preferably, the reactive oxygen
species is superoxide and hydrogen peroxide. More preferably, the
reactive oxygen species is hydrogen peroxide.
[0038] The term "resuscitation", as used herein, of injured
mammalian cells means the reversal of Cytotoxicity, the
stabilization of the cellular membrane, an increase in the
proliferation rate of the cell, and/or the normalization of
cellular functions such as the secretion of growth factors,
hormones, wound strength, texture, elasticity and pigmentation and
the like.
[0039] The term "therapeutically effective amount", as used herein,
means an amount of at least one compound of the invention, or a
pharmaceutically acceptable salt thereof, which is effective to
prevent, alleviate or ameliorate symptoms of disease or prolong the
survival of the subject being treated. Determination of a
therapeutically effective amount is within the skill in the
art.
[0040] The cells which may be treated with the therapeutic
wound-healing compositions of the present invention are mammalian
cells. Although the present therapeutic wound-healing compositions
are useful for treating mammalian epidermal keratinocytes and
mammalian monocytes, applicant contemplates that the therapeutic
wound-healing compositions may also be used to protect or
resuscitate all mammalian cells. Keratinocytes are representative
of normal mammalian cells and are the fastest proliferating cells
in the body. The correlation between the reaction of keratinocytes
to injury and therapy and that of mammalian cells in general is
very high. Monocytes are representative of specialized mammalian
cells such as the white blood cell in the immune system and the
organ cells in liver, kidney, heart, and brain. The mammalian cells
may be treated in vivo and in vitro.
[0041] Epidermal keratinocytes are the specialized epithelial cells
of the epidermis which synthesize keratin, a scleroprotein which is
the principal constituent of epidermis, hair, nails, horny tissue,
and the organic matrix of the enamel of teeth. Mammalian epidermal
keratinocytes constitute about 95% of the epidermal cells and
together with melanocytes form the binary system of the epidermis.
In its various successive stages, epidermal keratinocytes are also
known as basal cells, prickle cells, and granular cells.
[0042] Particular disease states to be treated in the invention
include wrinkles, aging skin, cellulitis, wounds and diseases which
are selected from the group consisting of infected and noninfected
wounds, bacterial infections, fungal infections, viral infections,
cold sores, thermal burns, sunburns, chemical burns, surgical
wounds, psoriasis, eczema, decubitus ulcers, cancer, diabetic
ulcers, dermatoses, inflammatory diseases, and wounds resulting
from laser treatment, micro-wounded skins, scratchs,
micro-needling, abrasions, laceration, puncture, dry skin, contact
with irritants, contact with allergens or other substances that are
harmful to skin or skin cells and any type of wound resulting from
laser treatments for the removal of scar and wrinkles, hair
removal.
[0043] The pyruvates are magnesium pyruvate and calcium pyruvate.
The total dosage of these two pyruvates may constitute about 0.10%
to about 5.0%, based on total weight of the composition. Preferred
is about 0.2% to about 4.0% and most preferred is about 0.5% to
about 4.0%. The dosage of L proline may constitute about 0.1% to
about 3.0%, based on total weight of the composition. Preferred is
about 0.2% to about 2.5% and most preferred is about 0.5% to about
2.5%. For the citrulline (L citrulline, citrulline malate or
combinations thereof) the ranges are the same as for L proline.
While the ranges are the same, in any given specific formula, the
amount of these two constituents may, and are likely, different
from each other, e.g., 2% L proline and 1.3% citrulline. The term
"liquid carrier" as used herein is meant to include any flowable
material that will transport the present invention formulation with
having detrimental effects thereon. These include water, oil,
water-based carriers, oil-based carriers, gels, emulsions, lotions,
creams, solutions and petrolatum. Preferred are oils, gels, lotions
and petrolatum. As shown elsewhere herein the forgoing formulas may
also include other actives without exceeding the scope of the
invention. These would include other pyruvates, acids, and
derivatives thereof, and/or antioxidants, antibacterial agents, and
other actives, as described below.
[0044] There may be pyruvate precursors included that may be
selected from the group consisting of pyruvyl-glycine,
pyruvyl-alanine, pyruvyl-cysteine, pyruvyl-leucine, pyruvyl-valine,
pyruvyl-isoleucine, pyruvyl-phenylalanine, pyruvamide, ethyl
pyruvate and salts of pyruvic acid. The pyruvate in the present
invention may be selected from the group consisting of pyruvic
acid, pharmaceutically acceptable esters such as methyl, ethyl, and
salts of pyruvic acid, prodrugs of pyruvic acid, and mixtures
thereof.
[0045] The alpha-keto acids having four or more carbon atoms may be
selected from the group consisting of oxaloacetic acid,
alpha-keto-glutaric acid, alpha-keto-butyric acid,
alpha-keto-adipic acid, .alpha.-keto-caproic acid, and
.alpha.-keto-isovaleric acid. The precursors of .alpha.-keto acids
having four or more carbon atoms may be selected from the group
consisting of .alpha.-keto acid-glycine, .alpha.-keto
acid-cysteine, .alpha.-keto acid-alanine, .alpha.-keto
acid-leucine, .alpha.-keto acid-valine, .alpha.-keto
acid-isoleucine, and .alpha.-keto acid-phenylalanine.
[0046] The amount of pyruvates or alpha keto acids in combination
with SRF present in the therapeutic wound healing compositions of
the present invention is a therapeutically effective amount. A
therapeutically effective amount of pyruvates and alpha keto acids
and SRF is that amount necessary for the inventive composition to
prevent and reduce injury to mammalian cells and increase the
synthesis of collagen, elastin, to increase wound strength texture
and pigmentation. The exact amount of pyruvates or alpha keto acids
and SRF is a matter of preference subject to such factors as the
type of condition being treated as well as the other ingredients in
the composition. In a preferred embodiment, pyruvates, or alpha
keto acids in combination with SRF is present in the therapeutic
wound healing composition in an amount from about 0.010% to about
5%. and more preferably from about 1% to about 3%, by weight.
[0047] In another preferred embodiment, the method may further
comprise contacting the mammalian cells with SRF and a therapeutic
agent. The therapeutic agent may be selected from the group
consisting of antibacterials, antivirals, antifungals, antitumors,
antihistamines, proteins, enzymes, hormones, nonsteroidal
anti-inflammatories, cytokines, and steroids. The therapeutic agent
may be administered prior to administration of SRF, or with the
administration of SRF.
[0048] The amount of therapeutic agent present in the therapeutic
compositions of the present invention is a therapeutically
effective amount. A therapeutically effective amount of a
therapeutic agent is the usual amount of therapeutic agent
necessary to treat the particular condition. The exact amount of
therapeutic agent is a matter of preference subject to such factors
as the type of condition being treated as well as the other
ingredients in the composition. In general, the amount of
antibacterial agent present is the ordinary dosage required to
obtain the desired result. Such dosages are known to the skilled
practitioner in the medical arts and are not a part of the present
invention.
[0049] The antibacterial agents which may be employed in the
therapeutic compositions may be selected from a wide variety of
water-soluble and water-insoluble drugs, and their acid addition or
metallic salts, useful for treating infections. Both organic and
inorganic salts may be used provided the antibacterial agent
maintains its medicament value. The antibacterial agents may be
selected from a wide range of therapeutic agents and mixtures of
therapeutic agents which may be administered in sustained release
or prolonged action form. Nonlimiting illustrative specific
examples of antibacterial agents include bismuth containing
compounds, sulfonamides; nitrofurans, metronidazole, tinidazole,
nimorazole, benzoic acid; aminoglycosides, macrolides, penicillins,
polypeptides, tetracyclines, cephalosporins, chloramphenicol, and
clindamycin. Preferably, the antibacterial agent is selected from
the group consisting of bismuth containing compounds, such as,
without limitation, bismuth aluminate, bismuth subcitrate, bismuth
subgalate, bismuth subsalicylate, and mixtures thereof; the
sulfonamides; the nitrofurans, such as nitrofurazone,
nitrofurantoin, and furozolidone; and miscellaneous antibacterials
such as metronidazole, tinidazole, nimorazole, and benzoic acid;
and antibiotics, including the aminoglycosides, such as gentamycin,
neomycin, kanamycin, and streptomycin; the macrolides, such as
erythromycin, clindamycin, and rifamycin; the penicillins, such as
penicillin G, penicillin V, Ampicillin and amoxicillin; the
polypeptides, such as bacitracin and polymyxin; the tetracyclines,
such as tetracycline, chlorotetracycline, oxytetracycline, and
doxycycline; the cephalosporins, such as cephalexin and
cephalothin; and miscellaneous antibiotics, such as
chloramphenicol, and clindamycin. More preferably, the
antibacterial agent is selected from the group consisting of
bismuth aluminate, bismuth subcitrate, bismuth subgalate, bismuth
subsalicylate, sulfonamides, nitrofurazone, nitrofurantoin,
furozolidone, metronidazole, tinidazole, nimorazole, benzoic acid,
gentamycin, neomycin, kanamycin, streptomycin, erythromycin,
clindamycin, rifamycin, penicillin G, penicillin V, Ampicillin
amoxicillin, bacitracin, polymyxin, tetracycline,
chlorotetracycline, oxytetracycline, doxycycline, cephalexin,
cephalothin, chloramphenicol, clindamycin and Mupericin.
[0050] The amount of antibacterial agent which may be employed in
the therapeutic compositions of the present invention may vary
depending upon the therapeutic dosage recommended or permitted for
the particular antibacterial agent. In general, the amount of
antibacterial agent present is the ordinary dosage required to
obtain the desired result. Such dosages are known to the skilled
practitioner in the medical arts and are not a part of the present
invention. In a preferred embodiment, the antibacterial agent in
the therapeutic composition is present in an amount from about
0.01% to about 10%, preferably from about 0.1% to about 5%, and
more preferably from about 1% to about 3%, by weight.
[0051] The antiviral agents which may be employed in the
therapeutic compositions may be selected from a wide variety of
water-soluble and water-insoluble drugs, and their acid addition or
metallic salts, useful for treating infections. Both organic and
inorganic salts may be used provided the antiviral agent maintains
its medicament value. The antiviral agents may be selected from a
wide range of therapeutic agents and mixtures of therapeutic agents
which may be administered in sustained release or prolonged action
form. Nonlimiting illustrative categories of such antiviral agents
include RNA synthesis inhibitors, protein synthesis inhibitors,
immunostimulating agents, protease inhibitors, and cytokines.
Nonlimiting illustrative specific examples of such antiviral agents
include the following medicaments.
[0052] Preferred antiviral agents to be employed may be selected
from the group consisting of acyclovir, Imiquimod, Foscarnet
sodium, ribavirin, vidarabine, ganeiclovir sodium, zidovudine,
phenol, amantadine hydrochloride, and interferon alpha.-n3. In a
preferred embodiment, the antiviral agent is selected from the
group consisting of acyclovir, foscarnet sodium, ribavirin,
vidarabine, Remesivir, and ganeiclovir sodium. In a more preferred
embodiment, the antiviral agent is acyclovir.
[0053] The amount of antiviral agent which may be employed in the
therapeutic compositions of the present invention may vary
depending upon the therapeutic dosage recommended or permitted for
the particular antiviral agent. In general, the amount of antiviral
agent present is the ordinary dosage required to obtain the desired
result. Such dosages are known to the skilled practitioner in the
medical arts and are not a part of the present invention. In a
preferred embodiment, the antiviral agent in the therapeutic
composition is present in an amount from about 0.1 to about 20%,
preferably from about 1 to about 10%, and more preferably from
about 2% to about 7%, by weight.
[0054] The antifungal agents which may be employed in the
therapeutic compositions may be selected from a wide variety of
water-soluble and water-insoluble drugs, and their acid addition or
metallic salts, useful for treating fungal infections. Both organic
and inorganic salts may be used provided the antifungal agent
maintains its medicament value. The antifungal agents may be
selected from a wide range of therapeutic agents and mixtures of
therapeutic agents which may be administered in sustained release
or prolonged action form. Nonlimiting illustrative specific
examples of antifungal agents include the following medicaments:
miconazole, clotrimazole, tioconazole, terconazole,
povidone-iodine, and butoconazole. Other antifungal agents are
lactic acid and sorbic acid. Preferred antifungal agents are
miconazole and clotrimazole.
[0055] The amount of antifungal agent which may be employed in the
therapeutic compositions of the present invention may vary
depending upon the therapeutic dosage recommended or permitted for
the particular antifungal agent. In general, the amount of
antifungal agent present is the ordinary dosage required to obtain
the desired result. Such dosages are known to the skilled
practitioner in the medical arts and are not a part of the present
invention. In a preferred embodiment, the antifungal agent in the
therapeutic composition is present in an amount from about 0.05% to
about 10%, preferably from about 0.1% to about 5%, and more
preferably from about 0.2% to about 4%, by weight.
[0056] The antitumor agents which may be employed in the
therapeutic compositions may be selected from a wide variety of
water-soluble and water-insoluble drugs, and their acid addition or
metallic salts, useful for treating cancers. Both organic and
inorganic salts may be used provided the antitumor agent maintains
its medicament value. The antitumor agents may be selected from a
wide range of therapeutic agents and mixtures of therapeutic agents
which may be administered in sustained release or prolonged action
form. Nonlimiting illustrative specific examples include
anti-metabolites, antibiotics, plant products, hormones, and other
miscellaneous chemotherapeutic agents. Chemically reactive drugs
having nonspecific action include alkylating agents and
N-alkyl-N-nitroso compounds. Examples of alkylating agents include
nitrogen mustards, azridines (ethylenimines), sulfonic acid esters,
and epoxides. Anti-metabolites are compounds that interfere with
the formation or utilization of a normal cellular metabolite and
include amino acid antagonists, 3, bromopyruvate, vitamin and
coenzyme antagonists, and antagonists of metabolites involved in
nucleic acid synthesis such as glutamine antagonists, folic acid
antagonists, pyrimidine antagonists, and purine antagonists.
[0057] Antibiotics are compounds produced by microorganisms that
have the ability to inhibit the growth of other organisms and
include actinomycins and related antibiotics, glutarimide
antibiotics, sarkomycin, fumagillin, streptonigrin, tenuazonic
acid, actinogan, peptinogan, and anthracyclic antibiotics such as
doxorubicin. Plant products include coichicine, podophyllotoxin,
and vinca alkaloids. Hormones include those steroids used in breast
and prostate cancer and corticosteroids used in leukemias and
lymphomas. Other miscellaneous chemotherapeutic agents include
urethan, hydroxyurea, and related compounds; thiosemicarbazones and
related compounds; phthalanilide and related compounds; and
triazenes and hydrazines, 2-Deoxy-D-glucose, Dichloroacetic acid,
often abbreviated DCA, is the chemical compound with formula
CHCl.sub.2COOH. It is an acid, an analogue of acetic acid,
Bromopyruvic acid, or bromopyruvate, is a synthetic brominated
derivative of pyruvic acid. The anticancer agent may also be a
monoclonal antibody or the use of X-rays. In a preferred
embodiment, the anticancer agent is an antibiotic. In a more
preferred embodiment, the anticancer agent is doxorubicin. In a
most preferred embodiment, the anticancer agent is doxorubicin.
[0058] Antioxidants are substances which inhibit oxidation or
suppress reactions promoted by oxygen or peroxides. Antioxidants,
especially lipid-soluble antioxidants, can be absorbed into the
cellular membrane to neutralize oxygen radicals and thereby protect
the membrane. The antioxidants useful in the present invention may
be selected from the group consisting of all forms of vitamin A
(retinol), all forms of vitamin B (3,4-didehydroretinol), all forms
of carotene such as .alpha.-carotene, .beta.-carotene,
gamma-carotene, .sigma.-carotene, all forms of vitamin C
(D-ascorbic acid, L-ascorbic acid), all forms of tocopherol such as
vitamin E (.alpha.-tocopherol,
3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltri-decyl)-2H-1-benzop-
-yran-6-ol), (.alpha.-tocopherol, gamma-tocopherol,
delta-tocopherol, tocoquinone, tocotrienol, and vitamin E esters
which readily undergo hydrolysis to vitamin E such as vitamin E
acetate and vitamin E succinate, and pharmaceutically acceptable
vitamin E salts such as vitamin E phosphate, pro-drugs of vitamin
A, carotene, vitamin C, and vitamin E, pharmaceutically acceptable
salts of vitamin A, carotene, vitamin C, .alpha.-lipoic acid and
vitamin E, and the like, and mixtures thereof. Preferably, the
antioxidant is selected from the group of lipid-soluble
antioxidants consisting of vitamin A, (carotene, vitamin E, vitamin
E acetate, N-acetylcysteine and mixtures thereof. More preferably,
the antioxidant is vitamin E or vitamin E acetate. Most preferably,
the antioxidant is vitamin E acetate.
[0059] The amount of antioxidant present in the therapeutic
wound-healing compositions of the present invention is also that
which is a therapeutically effective amount. A therapeutically
effective amount of antioxidant is that amount of antioxidant
necessary for the inventive composition to prevent and reduce
injury to mammalian cells or increase the resuscitation rate of
injured mammalian cells. The exact amount of antioxidant is a
matter of preference subject to such factors as the type of
condition being treated as well as the other ingredients in the
composition. In a preferred embodiment, the antioxidant is present
in the therapeutic wound-healing composition in an amount from
about 0.1% to about 40%, preferably from about 0.2% to about 30%,
and more preferably from about 0.5% to about 20%, by weight of the
therapeutic wound-healing composition.
[0060] The mixture of saturated and unsaturated fatty acids in the
present invention are those fatty acids required for the repair of
mammalian cellular membranes and the production of new cells. Fatty
acids are carboxylic acid compounds found in animal and vegetable
fat and oil. Fatty acids are classified as lipids and are composed
of chains of alkyl groups containing from 4 to 22 carbon atoms, 0-3
double bonds and characterized by a terminal carboxyl group,
--COOH. Fatty acids may be saturated or unsaturated and may be
solid, semisolid, or liquid. The most common saturated fatty acids
are butyric acid (C4), lauric acid (C12), palmitic acid (C16), and
stearic acid (C18). Unsaturated fatty acids are usually derived
from vegetables and consist of alkyl chains containing from 16 to
22 carbon atoms and 0-3 double bonds with the characteristic
terminal carboxyl group. The most common unsaturated fatty acids
are oleic acid, linoleic acid, and linolenic acid (all C18
acids).
[0061] In a preferred embodiment, the mixture of saturated and
unsaturated fatty acids has a composition similar to that of human
fat and comprises the following fatty acids: butyric acid, caproic
acid, caprylic acid, capric acid, lauric acid, myristic acid,
myristoleic acid, palmitic acid, palmitoleic acid, stearic acid,
oleic acid, linoleic acid, linolenic acid, arachidic acid, and
gadoleic acid. Preferably, butyric acid, caproic acid, caprylic
acid, capric acid, lauric acid, myristic acid, myristoleic acid,
palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic
acid, linolenic acid, arachidic acid, and gadoleic acid are present
in the mixture in about the following percentages by weight,
respectively (carbon chain number and number of unsaturation's are
shown parenthetically, respectively): 0.2%-0.4% (C4), 0.1% (C6),
0.3%-0.8% (C8), 2.2%-3.5% (C10), 0.9%-5.5% (C12), 2.8%-8.5% (C14),
0.1%-0.6% (C14:1), 23.2%-24.6% (C16), 1.8%-3.0% (C16:1), 6.9%-9.9%
(C18), 36.0%-36.5% (C18:1), 20%-20.6% (C18:2), 7.5%-7.8% (C18:3),
1.1%-4.9% (C20), and 3.3%-6.4% (C20:1).
[0062] In another preferred embodiment, the mixture of saturated
and unsaturated fatty acids comprises lecithin. Lecithin
(phosphatidylcholine) is a phosphatide found in all living
organisms (plants and animals) and is a significant constituent of
nervous tissue and brain substance. Lecithin is a mixture of the
diglycerides of stearic, palmitic, and oleic acids, linked to the
choline ester of phosphoric acid. The product of commerce is
predominantly soybean lecithin obtained as a by-product in the
manufacturing of soybean oil. Soybean Lecithin contains palmitic
acid 11.7%, stearic 4.0%, palmitoleic 8.6%, oleic 9.8%, linoleic
55.0%, linolenic 4.0%, C20 to C22 acids (includes arachidonic)
5.5%. Lecithin may be represented by the formula:
CH.sub.2OCOR--CHOCOR--CH.sub.2O--P(O).sub.2-OCH.sub.2CH.sub.2N+(CH.sub.3--
).sub.3, wherein R is selected from the group consisting of
stearic, palmitic, avocado oil and oleic acid.
[0063] The above fatty acids and percentages thereof present in the
fatty acid mixture are given as an example. The exact type of fatty
acid present in the fatty acid mixture and the exact amount of
fatty acid employed in the fatty acid mixture may be varied in
order to obtain the result desired in the final product and such
variations are now within the capabilities of those skilled in the
art without the need for undue experimentation.
[0064] The amount of fatty acids present in the therapeutic
wound-healing compositions of the present invention is a
therapeutically effective amount. A therapeutically effective
amount of fatty acids is that amount of fatty acids necessary to
prevent and reduce injury to mammalian cells or increase the
resuscitation rate of injured mammalian cells. The exact amount of
fatty acids employed is subject to such factors as the type and
distribution of fatty acids employed in the mixture, the type of
condition being treated, and the other ingredients in the
composition. In a preferred embodiment, the fatty acids are present
in the therapeutic wound-healing composition in an amount from
about 1.0% to about 50%, preferably from about 2.0% to about 45%,
and more preferably from about 2.5% to about 40%, by weight of the
therapeutic wound-healing composition.
EXAMPLES
[0065] The purpose of the examples below was to demonstrate
efficacy of (SRF) to repair and enhance cellular transport systems
to enhance the synthesis, secretion and transport of adhesion
molecules, skin phospholipids and type 1 collagen to increase wound
strength, wound texture, and pigmentation in wounds and treat
wrinkles. All other formulas to date without SRF, enhanced the rate
of healing, but did not increase wound strength, texture or
pigmentation. When the SRF formula was used, it produced superior
wound healing results. Magnesium pyruvate and calcium pyruvate are
needed by enzyme systems to increase the synthesis of adhesion
molecules and melanin. L proline increased collagen synthesis and
L-Citrulline increased the local production of nitric oxide that
increased vacuolization to enhance the rate of healing. In
combination they demonstrated a synergistic effect that increased
wound strength, texture and pigmentation over the individual
components.
Example 1 Rat Wound Strength Healing Studies
[0066] SRF with and without antioxidants and fatty acids,
administered topically, reversed impaired wound healing and
increased wound strength by doxorubicin (Doxo) and other cancer or
AIDS drugs in rats. Other cancer or HIV drugs tested that have been
shown to impair wound healing and decrease wound strength included
bleomycin, cisplatin, Urushiol, Imiquimod, Carboplatin,
floxuridine, methotrexate, oxaliplatin, Bevacizumab, radiation, and
the AIDS drugs including: Nucleoside Reverse Transcriptase
Inhibitors (NRTI), Fusion Inhibitors, Highly Active Antiretroviral
Therapy (HAART), and Non-Nucleoside Reverse Transcriptase
Inhibitors (NNRTI).
[0067] Rats given Doxo (6 mg/kg i.v.) were wounded with linear
dermal incisions, and the wound-breaking strength (WBS) was
compared among groups of rats differently treated with SRF (calcium
pyruvate, magnesium pyruvate, L proline and citrulline) with and
without antioxidants and fatty acids. Wound breaking strength is a
measurement of wound strength after injury compared to normal
noninjuried skin. Fourteen groups of five rats each were studied.
The SRF was administered topically daily. All groups were wounded
at postoperative day 0, at which time Doxo was given to all Groups.
All wounds were harvested at POD 21, and the mean wound breaking
strength (WBS) of each group was obtained using an Instron
Tensiometer. Doxorubicin impaired normal wound strength by 40
percent. Topical administration of SRF formulas restored WBS in
Doxo-treated rats to 86 percent of control values. In conclusion,
SRF was shown to restore wound strength, and texture which
demonstrates the increased synthesis and transport of collagen,
cardiolipin, phospholipids and elastin and pigmentation to nearly
normal levels in doxorubicin-impaired wounds.
[0068] See Table I:
TABLE-US-00001 TABLE I Testing The Various Topical Wound Healing
Formulas For Wound Breaking Strength And Texture And Pigmentation
Without And With SRF, Or With Or Without Certain SRF Components (2%
Calcium And 2% Magnesium Pyruvate, 1% L Proline And 1%
L-Citrulline) In The Topical Vehicles In Doxorubicin Treated
Wounded Hairless Rats Compared To Non-Wounded Normal Skin.
Percentage of Days to wound breaking Formula healing strength, by
day 21 Normal non wounded skin 0 100% Untreated control 18 40%
Vehicle Petrolatum 16 48% Calcium pyruvate without L proline 14 42%
Or L-citrulline Calcium pyruvate with L proline 16 44% Calcium
pyruvate with L-citrulline 16 51% Calcium pyruvate with L proline
13 54% And L-citrulline Magnesium pyruvate without L proline 14 44%
Or L-citrulline Magnesium pyruvate with L proline 15 42% Magnesium
pyruvate with L-citrulline 16 55% Magnesium pyruvate with L proline
13 58% And L-citrulline Magnesium pyruvate and 16 48% Calcium
pyruvate without L proline Or L-citrulline Calcium pyruvate and 13
52% Magnesium pyruvate alone With L-citrulline Calcium pyruvate and
Magnesium pyruvate with L Proline But without L-citrulline 15 42%
Calcium pyruvate and 11 88% Magnesium pyruvate with Proline And
with L-citrulline (SRF) Neosporin without SRF 15 51% Commercial
formula Neosporin commercial formula, 11 86% with SRF
[0069] All cancer drugs inhibit and damage mitochondria and impair
wound healing after treatment with chemotherapy or radiation. 1.6
days faster healing or greater than 10% over the vehicle control is
considered statistically significant by the FDA. Also, a 10%
increase in wound strength is considered clinically and
statistically significant by the FDA. Analysis of the biopsies
showed a greater increase in collagen type I, over type II or type
III with SRF. Type I is associated with wound strength. This
experiment clearly showed that the addition of SRF reversed the
negative effects of cancer drugs and enhanced the healing process
over other formula.
Example 2
[0070] All previous studies using pyruvate to treat wounds or be
used as an antioxidant used only the sodium salt form of pyruvic
acid. The drug products that were tested in previous studies used
1-3% sodium pyruvate and other sodium salts of alpha keto acids by
weight 1-3% concentrations in the lotions and triple antibiotic
formulations. Even though other salts of pyruvic acid like calcium
or magnesium etc. have been suggested, no one to date has evaluated
the different salts individually or in combination for their
ability to enhance cellular repair, mitochondrial repair, enhanced
synthesis of adhesion molecules and collagen, to increase wound
strength, texture and pigmentation, or to reduce irritation. Blood
levels of the various salts mg/liter are 3220 mg for Sodium, 200 mg
for potassium, 27 mg for magnesium, 70 mg for calcium, 1.1 mg for
zinc, 0.02 mg for manganese, 6 mg for lithium, 0.03 mg for
aluminum, 0.06 mg for ammonium and 0.36 mg for phosphorus. To
evaluate the irritation or toxicity levels of these salt
formulations, each salts of pyruvate (sodium, calcium, magnesium,
potassium, zinc, manganese, lithium and ammonium were tested. Using
the same concentrations of pyruvate of for each salt on skin laser
wounds in patients, the use of potassium pyruvate, magnesium
pyruvate, calcium pyruvate, zinc pyruvate, manganese pyruvate,
lithium pyruvate, aluminum pyruvate, and ammonium pyruvate were
tried. Potassium pyruvate, and calcium pyruvate, produced similar
wound healing results in patients as the sodium pyruvate
formulations. The zinc pyruvate, magnesium pyruvate, manganese
pyruvate, aluminum pyruvate, ammonium pyruvate, and lithium
pyruvate did not produce increases in healing over the base and did
not increase wound strength, texture or pigmentation to achieve the
results that SRF produced, and they were irritating and possibly
toxic producing skin irritation. When irritation and healing was
measured against formulas that contained Sodium pyruvate only, only
SRF was superior in reducing irritation from drugs by over 20%,
decreasing healing times by 25% and increasing wound strength by
45% which is considered clinically significant by the FDA. Table
I
[0071] The amount of the salts delivered in the formulations with
pyruvates were compared to the amounts of the salts in human blood.
The salts of pyruvic acid or pyruvate, sodium, potassium, and
calcium, were all under amounts found in human blood. The delivered
salts of pyruvic acid or pyruvate like including zinc in this
formulation was 840.times. blood levels, the magnesium was
40.times. blood levels, the manganese was 3,700.times. blood
levels, aluminum was 6100.times. times blood levels, the ammonium
was 533.times. times blood levels, and lithium was 50.times. blood
levels. The use of these salts of pyruvic acid at these levels
proved, that one cannot assume that all the salts of pyruvate are
non-irritating. Even though the use of the sodium salt of pyruvic
acid showed no irritation on dermabraided skin, and enhanced
healing, the others did produce irritation and the use of some of
these salts over time would have a negative effect on healing. The
sodium salt of pyruvate used was 59 times lower than sodium in
blood, the calcium was 1.5.times. times lower, and the potassium
was 3.2.times. times lower than blood levels, thus they were well
within the safe levels needed to deliver the pyruvate.
[0072] See table II
TABLE-US-00002 TABLE II Comparison Of Various Drugs Administered On
Wounds With Or Without Sodium Pyruvate Or SRF (2% Calcium & 2%
Magnesium Pyruvate, 1% L Proline And 1% L-Citrulline). Rating was
1-10, with 1 having the most amount of erythema and 10 having no
erythema. Wound healing was also measured in days to total
re-epithelization. Drug in Drugs in commercial Drug commercial
Drugs in Drug in formula commercial Drugs in formula commercial
commercial with sodium formula with commercial with sodium formula
formula pyruvate only. SRF. Various formula pyruvate only with SRF
Days to Days to Days to Drugs erythema erythema erythema healing
healing healing Mupirocin 4 6 8 20 15 11 Neosporin 5 5 6 20 13 11
erythromyci 6 5 8 19 13 9 mafenide 8 8 7 18 14 12 Acyclovir 4 5 7
18 13 10 Famciclovir 5 6 7 17 14 12 valaciclovir 8 8 8 18 17 14
Penciclovir 7 7 8 17 16 12 Chlotrimizo 4 6 8 22 15 11 butenafine 3
4 5 21 18 16 naftifine 3 6 7 20 14 13 econazole 6 6 6 19 17 14
ketoconazol 3 6 8 23 20 14 microazole 5 6 7 19 12 11 oxiconazole 6
7 9 18 14 11 tolnaftate 7 7 7 21 13 10 sulconazole 4 6 8 20 15 11
ciclopirox 4 5 9 20 11 9 nystatin 2 6 8 17 14 12 Retinol 1 3 7 19
15 9 Averages 4.6 5.4 7.05 18.5 16.05 11.6 The results clearly show
that SRF was superior in reducing erythema by 24% and decreasing
healing time over sodium pyruvate by 38% which is clinically
significant. indicates data missing or illegible when filed
Example 3 C-14 Testing
[0073] In rat tissue culture studies, using C 14 labeled calcium
and magnesium pyruvate C 14 labeled L-Proline and L-citrulline
(SRF); tissue culture analysis clearly showed that SRF increased
the production of collagen by 60% over the non SRF formulas which
only caused a 20% increase. All the individual 14c labeled
components including calcium and magnesium pyruvate, L-proline, and
L-citrulline only increased collagen by 10-18%. Incorporation of
[U-(14)C] L proline into total collagen increased 19-53% over the
2-hr with medium containing SRF. It was also discovered that 30% of
the radioactive calcium or magnesium pyruvate was incorporated into
phosphatidylcholine the main membrane cellular lipid (fatty acid),
and 10% into elastin, when calcium pyruvate, magnesium pyruvate
with L proline and citrulline, were present (SRF). In short,
calcium and magnesium are needed by the enzyme systems to
synthesize and transport cellular membrane components, and the
addition of L proline increased the synthesis of collagen. The use
of the SRF formulation was synergistic and produced the best
healing results of all previous combinations of alpha keto acids
tested to date and eliminated the need to provide fatty acids from
olive oil, cottonseed oil and cocoa butter.
Example 4 SRF Formulation
[0074] Topical formula: To one liter of lotion or gel vehicle or
petrolatum, add 0.7% (7.0 grams) to 3% (30 grams) of calcium
pyruvate, 1 gram or 0.1% to 2% of magnesium pyruvate and 0.1 to 1%
of L proline or and 0.1 to 1.0% L-citrulline. This formula has many
modifications to deliver the correct amount of calcium or magnesium
pyruvate, L proline and L-citrulline. Calcium can be delivered as
calcium pyruvate, with or without the addition of calcium chloride,
calcium carbonate, calcium acetate, calcium citrate, calcium
lactate, and calcium sulfate. Magnesium can be delivers as
magnesium pyruvate, with and without the addition of magnesium
chloride, magnesium phosphate, magnesium bicarbonate. L proline is
L-proline and citrulline as L-citrulline or citrulline malate. The
PH should be adjusted to 6.4 with sodium hydroxide. Pyruvate
provides the energy to synthesize membrane phospholipids, collagen
elastin and increase wound strength and texture. Calcium and
magnesium are needed for enzymes to make elastin and collagen.
Magnesium is also needed for mitochondrial membrane stability, for
the production of ATP and to increase wound strength and texture
and pigmentation. L-proline increases the synthesis and transport
of collagen to increase wound strength and citrulline increases
nitric oxide at the site to increase blood flow.
Example 4
[0075] The purpose of the study was to confirm the effects of
alpha-keto acid alone or in combinations for their effects on the
rate of cutaneous healing and wound breaking strength on 20
patients. The combinations were pyruvate and keto isovalerate and
pyruvate with alpha keto glutarate were tested with and without
SRF. The wound model utilized an arm design wherein the wound was
created by a well defined model of laser skin resurfacing a wound
that is very similar to a bad sunburn. 20 patients were tested with
various formula. Four laser wounds were made on each subject's arm
and the subjects were used as their own control. This model
utilized pulsed CO.sub.2 and Er: YAG lasers in a defined protocol
(computerized scanner for CO.sub.2 and uniform pulses with Er: YAG)
which removes the entire stratum. Patient diaries were maintained
to assess erythema, crusting, pain, itching, swelling, pigmentary
changes, and the day of first make-up application. Blinded
objective grading of improvement was independently assessed by 4
blinded observers at time intervals 3, 6, and 10 days, and 1, 2,
and 4 months. Chromometer measurements of erythema were also
analyzed and percentage moisture recorded.
[0076] Current laser skin resurfacing techniques averages 5-7 days
to skin re-epithelialization (healing) whereas a few years ago,
10-14 days was typical. For ethical reasons, the selected the
current "standard of care" model and thus the "delta value" for
wound-healing between active and control should be greater/more
significant for other types of wounds. All laser wounds remain red
for weeks to months, due to inflammation and the over production of
peroxynitrite. The study design did not address the final redness
issue in the diaries, but rather utilized a Minolta Chromometer to
look at relative values of erythema. The "LAB" color space model
was used wherein the "A" value correlates with erythema.
Reepithialization is the epithelial skin cells covering the entire
wound. Visual appearance is determined by how many days it takes a
wound to return to a normal appearance in terms of color (reduced
redness) pigmentation, and texture wound strength.
[0077] See table III and table IV
TABLE-US-00003 TABLE III Post Laser Healing Results.
Re-Epithelization with Various Formulations All in A Gel Based
Vehicle Delivered in Strips That Were Applied to One Of The Eight
Sites on An Arm Daily for Two Weeks. Pyruvate Is Sodium Pyruvate
and SRF Contains 2% Calcium and 2% Magnesium Pyruvate, 1% L Proline
And 1% L-Citrulline percent Days to Days to healing over healing
100% normal vehicle Formula reepithelization appearance control
untreated control 26 38 -- Gel lotion vehicle control 22 28 0%
Pyruvate & Ketoisovalerate 19 22 24% Without SRF Pyruvate &
ketoglutarate 20 24 22% Without SRF Pyruvate & Ketoisovalerate
18 20 23% And alpha ketoglutarate Without SRF SRF formula 15 14 56%
Pyruvate & ketoglutarate 15 15 59% With SRF formula Pyruvate
& ketoglutarate 11 14 57% & keto isovalerate in SRF formula
1.6 days faster healing or greater than 10% over the vehicle
control is considered statistically significant by the FDA.
TABLE-US-00004 TABLE IV Post Laser Healing Results,
Re-Epithelization With Various Formulations. Testing Of Formulas
Containing Sodium Pyruvate (Pyruvate) Other Keto Acids With And
Without Skin Resuscitation Factor (SRF) In A Petrolatum Base. Days
to percent Days to normal healing over Formula healing Appearance
vehicle control Untreated control 26 38 -- Aquaphor 22 29 0
Neosporin with sodium 16 24 26% Pyruvate, vitamin E and fatty acids
Pyruvate & Ketoisovalerate 15 21 22% Pyruvate &
ketoglutarate 15 21 24% Pyruvate & ketoglutarate 14 20 32%
Vitamin E and Fatty acids Pyruvate & keto isovalerate 13 19 33%
Vitamin E And fatty acids Sodium pyruvate, Ketoglutarate 12 18 76%
Vitamin E Fatty Acids and SRF SRF 12 18 74% Sodium Pyruvate,
vitamin 11 18 78% E and fatty acids in SRF formula
[0078] 1.6 days faster healing or greater than 10% over the vehicle
control is considered statistically significant by the FDA.
[0079] These experiments clearly showed that, SRF enhanced healing
by increasing the synthesis of cellular phospholipids, cellular
adhesions molecules, collagen, and increased wound healing, wound
strength, texture and pigmentation over all other formulas. The SRF
formula was also tested one hour after a total face laser
resurfacing treatment. The right side of the face was treated with
a cooling gel strip containing SRF to determine if the gel strip
with SRF could reduce erythema, pain redness and increase the rate
of healing over a non SRF treated site. The left side of the face
was treated with the gel strip without SRF. Two hours after
treatment, the right side treated with SRF produced a dramatic
reduction in pain, redness, swelling and increased cooling. The
left side of the face treated with the gel strip without SRF did
not reduce redness, pain, and swelling. Monitoring the effects of
SRF at the end of a week demonstrated that SRF produced better
textured skin and pigmentation tones. Over a three week course the
patients skin resolved and returned to normal in all places. The
gel strip contained 2% SRF by weight.
Example 6 Post Laser Treatment (4 Subjects
[0080] The purpose of this double blinded clinical study was to
evaluate if the addition of Skin Resuscitation Factor (SRF).TM.
technology would positively affect wound healing of two (2) test
products; 1) commercially available Abreva cream (docosonal) and 2)
a commercially available triple antibiotic petrolatum-based
ointment. The control products used in the study for comparison
purposes were identical to the commercial products except they did
not contain SRF.
[0081] Four laser wounds were made on each subject's arm and the
subjects were used as their own control. Although the study
population was small, the addition of SRF.TM. to both formulations
accelerated the re-epithelialization of the subjects' skin. The
relative increase among the subjects was a range of 10-60% at day 8
with some benefit evident at the remaining days until
re-epithelialization had occurred. These percentages are
statistically and clinically significant* values based on the
requirements for enhanced healing claims. (*Based per FDA, which
requires at least a 10% or approximately 1 day faster healing).
This experiment showed that these formulae healed laser treated
skin.
Example 7 (15 Subjects) Cold Sores
[0082] A commercial cold sore formula was purchased and the
enumerated keto acids were placed into it to treat cold sores, both
as a combination and singly as well. The commercial formulation by
itself was utilized as a control. The normal cold sore formulas
with phenol, an antiviral agent, did not work very well. However,
when SRF was placed in the cold sore formulation both with and
without anti-oxidants and fatty acids, they heal the cold sore at a
much faster rate 10-40% when tested on a cold sore sufferer. The
same experiment was done with virally infected cells and the
combination of pyruvate and .alpha.-Ketoisovalerate with SRF
decreased viral plaque formation by 50%. Viral plaques are a direct
measure of viral numbers in infected cells. The antiviral drug,
Acyclovir also decreased viral plaques by 60% and SRF in
combination with acyclovir, totally eliminated the virus from the
infected cells. Other antiviral agents like docosonal produced
better efficacy with the addition of SRF. Other antivirals were
evaluated with SRF to determine if the addition of SRF could reduce
the skin irritation produced by Foscarnet sodium, Ribavirin,
imiquimod, vidarabine, ganciclovir sodium, zidovudine, phenol,
amantadine hydrochloride, and interferon. alpha.-n3. Even though
some of these anti virals are not effective against a cold sore,
they were evaluated on human cold sores to determine if SRF could
reduce the irritation and enhance healing at the infection site.
All formula with SRF healed cold sore two days faster than
non-treatments and when this formula was combined with antiviral,
like acyclovir or docosonal, the viral lesions healed 1.5 days
faster than the commercial antiviral agent alone (Abreva or
acyclovir). The patients rated all the SRF antiviral formulations
higher than the formulations containing the anti virals alone. On a
1-10 scale with 1 being far worse irritating than the formulation
without SRF and 5 being equal and 10 being the least irritating
most of the SRF formulations received a 7-8 compared to the
formulations with only the antivirals (4-5 rating). The use of SRF
formula with the addition of 0.1% Zinc lysine formula, produced the
best lesion healing results.
Example 8 Anti-Wrinkle, Stretch Marks and Scar Removal and
Prevention in Acne Tissue Culture Analysis of Collagen Production
with SRF and Alpha Ketoglutarate Acid
[0083] Alpha-ketoglutarate SRF formula worked synergistically to
stimulated procollagen production in cultured human dermal
fibroblasts, and decreases UVB-induced wrinkle formation in humans.
The single salt of sodium alpha ketoglutarate or in combination
with sodium pyruvate, was not as effective. Alpha-ketoglutarate is
a key intermediate in the Krebs cycle, and a rate-limiting cofactor
of prolyl-4-hydroxylase. It also has a potent effect on increasing
the proline pool during collagen production. To investigate the
effects of alpha-ketoglutarate on procollagen production and
wrinkle formation, we conducted experiments in cultured human
dermal fibroblasts and UVB-irradiated hairless mice. Based on ELISA
measurements, alpha-ketoglutarate (10 microM) stimulated
procollagen production in fibroblasts by 25.6+/-4.6% compared to
vehicle (dH(2)O)-treated control cells. Also, we demonstrated that
alpha-ketoglutarate increased activities of prolidase, which is
known to play an important role in collagen metabolism, in
fibroblasts and N-benzyloxycarbonyl-L-proline (Cbz-Pro), prolidase
inhibitor, inhibited procollagen synthesis by alpha-ketoglutarate
in fibroblasts. To determine the effect of topically applied
alpha-ketoglutarate on wrinkle formation, alpha-ketoglutarate (1%)
and vehicle (70% propylene glycol, 30% ethanol) were applied on the
skin of UVB-induced patients 6 weeks. We found that
alpha-ketoglutarate decreased wrinkle formation upon long-term
topical application, but was irritating. These results suggest that
alpha-ketoglutarate diminishes UVB-induced wrinkle formation by
increasing collagen production, through a pathway that involves
prolidase activation. Therefore, application of alpha-ketoglutarate
may represent an effective anti-wrinkle agent for the cosmetic
field. For example, alpha-keto glutarate reacts with hydrogen
peroxide to form succinic acid which increases the production of
hydrogen peroxide. Thus, when used alone as the sodium salt, with
vitamin E and fatty acids, it is irritating and can injure cells
and mitochondria and increase the damage to cellular membranes and
inhibit the synthesis of collagen, elastin and decrease cellular
longevity. Its use in this manner has been shown to delay healing
and is irritating. Alpha keto glutarate in combination with SRF
enhanced collagen deposition and wound strength with no skin
irritation.
[0084] Skin application of SRF to remove wrinkles and firm skin
with Collagen induction therapy (CIT) also known as microneedling
RF or skin needling is a cosmetic procedure that involves
repeatedly puncturing the skin with tiny, sterile needles
(microneedling the skin). CIT should be separated from other
contexts in which microneedling devices are used on the skin, e.g.
transdermal drug delivery, vaccination. It is a technique for which
research is ongoing but has been used for a number of skin problems
including scarring and acne. Platelet-rich plasma (PRP) can be
combined with collagen induction therapy treatment to enhance
results. PRP is derived from the patient's own blood and contains
many growth factors that increase collagen production. It can be
applied topically to the entire treatment area during and after
collagen induction therapy treatments and/or injected intradermally
to scars. The use of microneedling followed by the application of
SRF increased collagen deposition 78% more than microneedled skin
alone. It also was used on turkey necks and wrinkles.
Example 9 Retinol Clinical Evaluation
[0085] In this randomized, parallel, double-blind, controlled
clinical study, women applied to the entire face for 3 months in
the morning and in the evening either the Retinol 0.2% cream or
Retinol 0.2% cream with SRF (2% of calcium pyruvate, 2% of
magnesium pyruvate, 1% of L proline, and 1% of L citrulline).
Clinical and instrumental parameters were assessed at days 0, 4,
28, 56, and 84. Subject perception of the efficacy, tolerance of
the tested products was assessed at days 1, 4, 10, 28, 56, and 84.
A total of 34 women were enrolled, 17 that used the Retinol 0.2%
cream and 17 that used the retinol 0.2% cream with SRF. Both
products improved wrinkles, mottled pigmentation, pores, and global
photodamage. A statistically significant differences was noted
between Retinol 0.2% cream and Retinol 0.2% SRF cream. Adverse
effects were mostly graded mild. Overall, Retinol 0.2%/with SRF
cream was better tolerated than Retinol 0.2% cream without SRF. The
Retinol cream without SRF was associated with a certain number of
adverse effects (pruritus, burning sensation, erythema, and
desquamation) currently known as "retinoid reaction". These adverse
effects were totally eliminated when retinol was combined with
SRF.
[0086] Clinical efficacy evaluations of wrinkles (frontal and
glabellar area, eyes area, nasolabial fold), pores, and
pigmentation were performed by a dermatologist using the photo
numeric scales for aged skin before the first product application
on Day 0 (baseline) and then on Day 4, Day 10, Day 28, Day 56, and
Day 84. The Dermascore was used to assess, at all study visits,
changes in pores and pigmentation. The Dermascore device is a
modified dermatoscope equipped with appropriate polarizing filters.
The color aspect alone is observed by removing the specular
component with a cross-polarized analyzer, whereas the pore
visibility is strengthened by increasing the proportion of specular
reflexion with a parallel polarized analyzer. Mean values of
roughness, depth, and texture were calculated from parameters
measured along 16 lines around a defined point of the area to be
evaluated. Subjects remained for at least 30 min prior and during
instrumental measurements in a room with controlled temperature
(20.degree. C..+-.2.degree. C.) and a relative humidity of
50%.+-.5%. A decrease of values indicated an improvement of the
skin relief.
[0087] Clinical Assessments
[0088] After 4 days of treatment with Retinol with SRF, a
statistically significant improvement over retinol treatment
without SRF was observed for wrinkles (P<0.001) nasolabial fold
(p<0.001), and pigmentation (P<0.005) These differences were
sustained from Day 5 onwards for wrinkles and from Day 8 onwards
for pores and pigmentation. Improvement of horizontal wrinkles on
the forehead and crow's feet was statistically significant over the
Retinol cream with out SRF. A significant improvement was observed
with Retinol 0.2% with SRF for the roughness, depth and texture as
early as Day 8 vs day 28 for the Retinol cream without SRF.
Example 10 Post Laser Treatment (37 Subjects) for Tattoo and Hair
Removal
[0089] SRF applied in a gel on post laser treatments for hair or
tattoo removal decreased the irritation form the laser treatment
and increased the healing time form 16 days to 10 days.
Example 11 Treatment of Infected Wounds
[0090] In this experiment a triple antibiotic formulation with SRF
was used to treat skin infections and compared to a standard triple
antibiotic without SRF in five patients. The patients had multiple
infection sites and each site was treated with one of the two
formulations. In all cases the infections resolved and were
eliminated by day 7 with the SRF formulation vs the triple
antibiotic without SRF which took 9 days to resolve. See FIG. 1.
Other antibiotics tested in this manner included, Mupericin,
neomycin bacitracin, polymyxin kanamycin, streptomycin,
erythromycin, clindamycin, penicillin G, penicillin V, Ampicillin
amoxicillin, tetracycline, chlortetracycline, oxytetracycline,
doxycycline, cephalexin, cephalothin, chloramphenicol, and
clindamycin, and several others.
[0091] As shown the triple antibiotic, Neosporin with SRF (2% of
calcium pyruvate, 2% of magnesium pyruvate, 1% of L proline, and 1%
of L citrulline) (D, Right Column) healed 2-3 days faster than the
triple antibiotic without SRF Neosporin (C, left column) in
infected wounds. Formula (D) contained SRF, in a triple antibiotic
formula Neosporin.
Example 12 Fungal Infected Wounds
[0092] In this experiment, various antifungals were tested for
their ability to kill an infection. The antifungal agents included
the following medicaments: miconazole, chlotrimizole, tioconazole,
terconazole, povidone-iodine, and butoconazole. In these
experiments, SRF was placed into commercial antifungals
formulations and were evaluated for enhanced efficacy. The patients
reported that their fungal infections, including athlete's foot,
jock itch and some skin infections cleared faster than their use of
the commercial formulation.
Example 13. Treatment of Decubitus, Pressure Sores, Venous Status
Ulcers and Wounds that do not Heal
[0093] The reason wounds do not heal is due to many reasons like
underlying disease states, poor nutrition and age. Protein
deficiency impairs wound healing since protein is needed for
fibroblast proliferation, new blood vessel formation, and collagen
production. Protein deficiency leads to impaired healing through
impaired collagen synthesis and deposition, decreased skin and
facial wound-tensile strength. In addition to the clear role of
protein intake in wound healing, proteins are essential to function
of the immune system.
[0094] To the SRF formula of containing zinc pyruvate we added
Arginine which is an essential amino acid and is involved in
collagen synthesis of the healing wounds. At times of stress or
injury, arginine synthesis is insufficient to meet the demands of
increased protein turnover and the body arginine stores decrease
rapidly. It is during these times that arginine becomes an
indispensable amino acid in the process of wound healing. Like
arginine, calcium pyruvate increases the synthesis of Nitric Oxide
and cytokines needed to kill bacterial and virally infected cells.
Nitric oxide is produced by the human body to kill infections. The
beneficial effects of Nitric Oxide on wound repair may be its
functional influences on angiogenesis, inflammation, cell
proliferation, matrix deposition, and remodeling. Nitric oxide
levels were increased by 42% at wound sites when SRF was applied.
When tested on non-healing wounds, on various patients, the formula
enhanced healing allowing all 6 patients to achieve complete
healing of the nonhealing wound in 14-28 days.
Example 14 Eczema and Psoriasis
[0095] Ten patients with varying forms of Eczema were given an
emollient cream with the SRF formula, containing 2% colloid oatmeal
to test for one month. These patients used various forms of
products and were asked to compare the SRF formula to their current
product. 9 out of 10 patients rated the SRF formula superior to
their current product including products with steroids. We also
purchased the Neosporin essentials products used to treat eczema.
We added the SRF (2% of calcium pyruvate, 2% of magnesium pyruvate,
1% of L proline, and 1% of L citrulline) formula to the two
products, the daily moisturizing cream with colloidal oatmeal and
to their 1% hydrocortisone anti-itch formula. The patients were
asked to rate the products after seven days of use. They tested the
non modified Neosporin products on two of five locations on the
same arm. The other two spots were used to test the modified SRF
product and one was used as an untreated control. The four
locations were picked randomly and the test products were blinded.
Each patient served as their own control. The results were
evaluated on a 1-10 scale for resolution of the symptoms by the
patient and the expert evaluator. The results were tabulated for
all 10 patients in table 5 as percentage of reduction of
symptoms.
[0096] See table V.
TABLE-US-00005 TABLE V Reduction Of Itching, Skin Irritation,
Inflammation & Rashes With And Without SRF. % % % Reduction
Reduction % Reduction of skin of inflam- Reduction Formula of
itching irritation mation of rash Untreated control 10% 5% 0 0
Neosporin Oatmeal 20% 30% 25% 29% Non modified Neosporin Oatmeal
60% 70% 55% 60% With SRF Neosporin Hydrocorti- 70% 75% 80% 80% sone
Nonmodified Neosporin Hydrocorti- 90% 92% 100% 96% sone With
SRF
[0097] Topical antihistamines and topical steroids have been used
for years to reduce skin inflammation. The problem is that they do
not improve healing and, in some cases, prolong the healing process
of injured skin. The average days to healing with these products is
around 18 days. The addition of SRF to these commercial creams or
lotions decreased the days to healing to 12 days a 34% increase in
healing. The antihistamines tested were diphenhydramine, doxepin,
and the topical steroids tested were aclometasone, fluocinolone,
hydrocortisone, desonide, mometasone, prednicarbate, trimcinalone,
amcinonide, diflorasone, betamethasone and clobetasol. Patients
with severe psoriasis requiring the use of immunosuppressant's were
treated topically with SRF that contained 3-Bromopyruvate which
produced dramatic results in resolving severe psoriasis.
[0098] SRF increases the synthesis of Nitric Oxide (NO) to enhance
the rate of healing and kill infections. Nitric oxide is released
in the skin after exposure to sunlight and this molecule can help
reduce the inflammation. This in turn leads to reduction of the
itching symptoms that leads to such distress among the persons
suffering from eczema. Several techniques can be used to measure
nitric oxide in intact skin. The intracellular nitric oxide assay
uses a fluorescent probe for direct nitric oxide detection in live
intact cells. The nitric oxide-specific probe is extremely
photostable and provides a wide pH stability range of 5.5 to 9.0.
Also, an invitro nitric oxide assay measures nitric oxide
indirectly via the breakdown products of nitric oxide (nitrite and
nitrate) in cell lysates, tissue homogenates, plasma, serum,
saliva, urine, and cell culture supernatants. Assays are available
for either colorimetric or fluorometric detection in a 96-well
plate.
[0099] For this study the team of researchers exposed some healthy
volunteer participants to SRF, on a small patch of eczema skin. On
testing they noted presence of nitric oxide in their blood stream
as a result of this exposure and an increase in skin nitric Oxide.
Mean basal NO concentration measured in vivo, in twenty-one
dialysis probes in twenty-one subjects were 0-49+0 06/M. Following
intradermal injection of Pyruvate, NO concentration in the
dialysate from the weal response increased to a maximum of
1*68+0-16/LM within 2 min (P<0.0002). Following intradermal
injection of SRF, NO concentration in the dialysate from the weal
response increased to a maximum of 2*38+0-16/LM within 2 min
(P<0.0002). In the flare area, NO levels rose to 1 19+0-26/SM
(P<0.018) at 6-8 min after SRF injection, the maximum level
coinciding with the flare front crossing the dialysis probe. The
mean areas of the weal and flare responses were 2-3+0 3 cm2 and 19
5+1-7 cm2 (n=6) respectively. No significant increase in NO was
detected following intradermal saline.
[0100] Nitric oxide in turn was noted to activate some specialized
immune cells called the regulatory T cells. These cells kill the
hyperactive inflammatory responses that lead to the symptoms of
eczema in the first place. Nitric Oxide also turns off N F kappa B
the major inflammatory gene in humans. NO is an important regulator
and mediator of numerous processes in the nervous, immune, and
cardiovascular systems. These include vascular smooth muscle
relaxation, resulting in arterial vasodilation and increasing blood
flow. NO is also a neurotransmitter and has been associated with
neuronal activity. NO also partially mediates macrophage
cytotoxicity against microbes and tumor cells.
TABLE-US-00006 TABLE VI Testing various topical wound healing
formulas for production of Nitric oxide (NO) and days to healing
wounds with SRF or without certain SRF components (2% calcium and
2% magnesium pyruvate, 1% L proline and 1% L-citrulline) in wounded
hairless Rats compared to non-wounded normal skin. % of NO levels
Days to against normal Formula healing nonwounded skin Normal non
wounded skin 0 100% Untreated control 19 20% Vehicle Petrolatum 16
18% Calcium pyruvate without L proline 15 22% Or L-citrulline
Calcium pyruvate with L proline 16 23% Calcium pyruvate with
L-citrulline 15 31% Calcium pyruvate with L proline 14 34% And
L-citrulline Magnesium pyruvate without L proline 13 36% Or
L-citrulline Magnesium pyruvate with L proline 15 38% Magnesium
pyruvate with L-citrulline 16 45% Magnesium pyruvate with L proline
14 51% And L-citrulline Magnesium pyruvate and 16 46% Calcium
pyruvate without L proline Or L-citrulline Calcium pyruvate and 13
52% Magnesium pyruvate alone With L-citrulline Calcium pyruvate and
15 37% Magnesium pyruvate with L Proline But without L-citrulline
Calcium pyruvate and 11 88% Magnesium pyruvate with L- Proline and
with L-citrulline (SRF)
Example 15
[0101] Various commercial lotions and creams were purchased to
evaluate these formulas with and without SRF in patients that
purchased them. The products included Gold Bond pain and itch
relief cream, Gold bond ultimate skin protectant lotion, Gold bond
ultimate restoring skin therapy lotion, gold bond ultimate
softening lotion, and Gold bond ultimate healing Aloe skin therapy
lotion and Gold Bond eczema formula. 143 patients were asked to
evaluate the modified formula with SRF on a 1-10 scale, with 1
being worse than their current product, 5 being equal and 10 being
far superior to the current product without SRF. The patients used
the modified SRF product for one month and 109 returned the
questionnaire with an overall rating of a 7.8 for the SRF
containing products with an overall rating of 8.2 for likely
purchase, compared to the rating of 4-6 for the other products
without SRF.
Example 16 Pigmentation Study
[0102] Six laser wounds were made on the left arm. As an example,
Mupirocin was placed on the first wound site, Mupirocin plus sodium
pyruvate was placed on the second wound site and Mupirocin with SRF
was placed on third wound site. The same process was repeated with
another drug for sites 4-6. Every two days the bandages coated with
the drugs were removed and replaced five times. Pigmentation
measurements range from 1-10 with 10 being totally normal. The
mupirocin drug formulas averaged a 4.9 for pigmentation. The
addition of sodium pyruvate raised that to 6.0, an increase of 18%
for pigmentation. The addition of the SRF averaged 7.45 a 34%
increase in pigmentation.
Example 17: Cancer Drugs Damage Normal Cells and Organs and Inhibit
the Synthesis of Nitric Oxide
[0103] The use of SRF offers promise in battling cancer-related
deaths in the United States by decreasing hypoxemia at cancer sites
and increasing the synthesis of nitric oxide to enhance the
cellular death of cancer with and without cancer drugs.
[0104] Hypoxia within regions of solid tumors including lung
cancers, is associated with resistance to standard treatments,
particularly radiotherapy. Conventional drug therapy, which depends
on reaching the cancer through the bloodstream, can be less
effective in hypoxic tumors. Low oxygen levels in a cell interrupt
the activity of oxidative phosphorylation, a term for the highly
efficient way that cells normally use to convert food to energy. As
oxygen decreases, the cells switch to glycolysis to produce their
energy units, called ATP. Glycolysis is a drastically less
efficient way to obtain energy, and so the cancer cells must work
even harder to obtain even more food, specifically glucose, to
survive. When oxygen levels dip dangerously low, angiogenesis, or
the process of creating new blood vessels, begins. The new blood
vessels provide fresh oxygen, thus improving oxygen levels in the
cell and tumor and slowing the cancer growth--but only
temporarily.
[0105] Drug designers have taken advantage of the hypoxic regions
in tumors and designed anticancer drugs that are specifically
active or activated under hypoxic conditions. For example,
hypoxia-activated prodrugs like 3-bromopyruvate, are chemically
modified to be inactive, but when administered to the body and
exposed to hypoxic conditions (such as in a tumor including skin
cancer), they are metabolized or otherwise converted into the
active, anticancer form. Despite these new drugs, there is an
ongoing need for innovative approaches to anticancer therapy.
Example 18: Pretreatment of Normal Cells Co Cultured with Cancer
Cells, Followed by Treatment with Doxorubicin
[0106] Peripheral blood monocytes and U937 monocytic leukemia tumor
cells were placed in sterile culture flasks and maintained in
culture using Dulbecco's Minimal Essential Medium, with 10% fetal
calf serum, supplemented with 2 mM glutamine and Pen/Strep. The
cytotoxicity of the cytotoxic agent on the cells was analyzed by
propidium iodide exclusion techniques and flow cytometric
quantitation. Viability of the cells was quantified as the number
of cells that excluded the vital dye trypan blue. SRF was dissolved
in distilled water and the solution was adjusted to pH 7.4.
Solutions were sterile filtered. Stock solutions were prepared so
that the vehicle would not be more than 1% of the total volume of
the culture media. In H-Thymidine Radiosotopic Incorporation
Measurement of cytotoxicity, SRF was examined for its ability to
decrease the cytotoxicity of Doxorubicin to U937 monocytic leukemia
cells and normal peripheral blood monocytes. The optimal
concentrations of the agents that were able to protect cells
against Doxorubicin induced cytotoxicity were the 0.1% calcium
pyruvate, 0.1% magnesium pyruvate, 0.1% L-proline, and 0.1%
L-citrulline (SRF). Susceptibility studies were conducted to
determine the optimal treatment time of the cells with the
cryoprotective agents prior to treatment of the cells with the
cytotoxic agent. The normal cells and U937 leukemic tumor cells
were pretreated separately in "wash out" studies with the single
agents alone, and in combination, at the optimal concentration
described above for various time periods, washed with fresh medium
to remove the agents, and treated with the cytotoxic agent. The
co-culture of normal and U937 leukemic minor cells was treated
essentially in the same manner except that the cells were not
treated separately, but co-cultured. The optimal pretreatment time
of the cells with SRF found to be 24 hours prior to treatment of
the cells with Doxorubicin. The cells were then placed in culture
medium without the protective agents. The length of time that the
cryoprotection lasted was 24 hours following Doxorubicin treatment.
At this time, peripheral cell viability is a limiting factor
because these cells are normal cells and do not remain in culture
for extended periods of time.
[0107] Normal and U937 tumor cells were co-cultured and the
cytotoxicity of Doxorubicin on the cells was determined by
viability assays which examined the differential ability of the
cytoprotective compositions alone, and in combinations, to protect
the normal cells from the cytotoxicity of the chemotherapeutic
agent.
[0108] The cells were isolated and examined for morphological
evidence of cytotoxicity or prevention of cytotoxicity. These
studies determined the cytoprotective effect of SRF on the normal
and tumor cells. DNA synthesis studies using 3H-thymidine (1
uCi/well) were carried out 4 hours prior to termination of the
experiment to determine the effect of the formulations on the
proliferation of the cells as a measure of the prevention of
cytotoxicity and the extent of Doxorubicin-induced cytotoxicity.
Propidium iodide exclusion analysis was carried out for direct
quantitation of the cytotoxicity and the prevention of
cytotoxicity. Each set of studies was performed in triplicate so
that statistical analysis of the significant differences between
the treatment groups could be conducted.
[0109] SRF provided significant protection to the normal peripheral
monocytes and did not protect the tumor cells from the effects of
the Cytotoxic agent. Wash-out studies were conducted to determine
viability of the peripheral blood monocytes co-cultured with U937
monocytic leukemia cells after 24 hour pretreatment of the cells
with SRF, which is also a mitochondrial protective agent, followed
by administration of Doxorubicin. The viability of the control
normal peripheral cells was enhanced from 55% to 68% with the use
of SRF, whereas the viability of the control U937 cells was
decreased from 43% to 12%. Thus the use of SRF protected normal
cells for 24 hours, while the leukemia cells died.
[0110] See table VIII.
TABLE-US-00007 TABLE VIII Comparison of various cancer drugs in
various media. Percentage of viable non-cancerous cells after
incubation with anticancer drugs Drugs in Drugs in Percentage
commercial commercial of viable formula Drugs in formula cells
after Drugs in sodium commercial with SRF Various incubation
commercial pyruvate formula repeat Drugs with drugs formula without
SRF with SRF study anastrozole 24 25 50 86 94 Bleomycin 31 33 47 98
98 cisplatin 46 43 58 88 91 Carboplatin 42 50 56 87 97 floxuridine
38 38 58 92 91 methotrexate 42 43 68 94 98 oxaliplatin 23 24 41 95
98 Bevacizumab 21 27 40 88 87
[0111] Other drugs tested produced similar results as stated in the
above table. They included Crizotinib, Docetaxel, Erlotinib,
Etoposide, Gemcitabine, Irinotecan, Paclitaxel, Pemetrexed,
Vinorelbine.
Example 19: Treatment of Patients with Various Cancers
[0112] To date most patients with cancer that are treated with
radiation or cancer drugs show a partial reduction of tumor sizes,
but in most cases the cancer remains and life expectances increases
for a short period of time. This is the case for tumors. Five
patients with various cancers were treated with 3-Bromopyruvate, an
alkylating agent and a well-known inhibitor of energy metabolism.
Results to date have been mixed. The problem with most cancer drugs
is not only their toxicity to noncancerous tissue, but is their
inability to completely eradicate the cancer. Cancer drugs cause
hypoxia in normal noncancerous cells and they destroy tissue and
their ability to heal. In mice studies conducted by others, they
investigated the chemo preventive activity of 3-bromopyruvate. For
the topical treatment the mice were treated with 10-30 mg/5 ml
daily and treated for 8 weeks. 3-bromopyruvate significantly
decreased tumor multiplicity and tumor load by 40% and 60%,
respectively, at a dose of 10 mg/5 mL. Treatment with
3-bromopyruvate in tissue cultures of cancer cells showed an
increased immune histo chemical staining for cleaved caspase-3,
suggesting that the tumor inhibitory effects of 3-bromopyruvate
were through induction of apoptosis. 3-Bromopyruvate also
dissociated hexokinase II from mitochondria, reduced hexokinase
activity, and blocked energy metabolism in cancer cells, finally
triggered cancer cell death and induced apoptosis through
caspase-3, and PARP in human lung cancer cell line. The problem
with 3-bromopyruvate was its toxicity to normal cells. The formula
needed to protect noncancerous cells from 3-bromopyruvate and to
enhance its effect on tumors, was the addition of SRF 1-2 hours
prior to the administration of the 10 mg of 3-bromopyruvate. The
investigators were given SRF formula 1-2 hours prior the treatment
of the cancer drug. This approach reduced tumor sizes by 90%
compared the 60% reduction with 3 bromopyruvate by itself. The
addition of dichloroacetate with 3-bromopyruvate to the SRF formula
was the best formula decreasing tumor loads by 95%, especially with
the addition of magnesium bicarbonate that increased the PH to 7.9
to neutralize the lactic acid produced in tumors that enhance
tumorigenesis.
Example 21: Cancer Trial Pilot Study
[0113] In eleven patients with various cancers, were given The SRF,
which increased Nitric oxide levels in these patients to enhance
the effect of the chemotherapy or radiation, and protected normal
cells and help reduce hypoxia in cancer cells.
[0114] These patients applied the SRF to the topical cancer or
injected it into the tumors. one to two hours before they were
treated with cancer drugs or radiation, given by current standard
methods. These patients continued chemo therapy beyond the eight
weeks and ten patients are still alive six to 10 years later,
beating the national survival rate by 4 years.
[0115] Other drugs tested given by IV or oral or topically,
standard treatments reduces tumor sizes or loads by an average
29.2% as sited in the literature. These drugs were Crizotinib,
Docetaxel, Erlotinib, Etoposide, Gemcitabine, Irinotecan,
Paclitaxel, Pemetrexed, Vinorelbine.
Example 22 Tissue Culture Studies of Herpes Simplex HSV-1 or 2 or
Human Papillomavirus HPV
[0116] To investigate the ability of SRF to regulate the
inflammatory process during an infection, the MatTek EpiDerm Assay
was used. The MatTek Epiderm tissue samples were treated with SRF
and the combination with immunostimulators Imiquimod and antiviral
agents Acyclovir determine if the combination would regulate nitric
oxide, IL-1 and IL-8 up or down during a simulated infection.
Following a one-hour equilibration, the Epiderm tissues were placed
into the incubator (37.degree. C., 5% CO.sub.2) in assay medium.
The old medium was replaced with fresh medium and the test articles
were applied to the tissue samples. The test articles remained in
contact with the tissue for various dosing times, one hour, then at
four hours, and at 20 hours. The testing was run in duplicate.
Various immunostimulators Imiquimod or sodium dodecyl sulfate
(SDS), glycoprotein D (gpD) were used singly or with the SRF to
replicate an infection, along with vehicle controls. Untreated
samples were used as negative controls. Following treatment, the
media from the tissue samples were tested in Elisa kits for IL-1
and IL-8 according to the manufacture's protocols.
[0117] Results: The primary end points were the levels of IL-8 and
IL-1 after treatment with an immunostimulators, and SRF. The
immunostimulators did not increase the cytokines by themselves.
This model did not have white blood cells to respond to the
immunostimulators or produce oxygen radicals. The immunostimulators
in combination with SRF increased Nitric oxide, and IL-8 over 400%,
which shows direct antimicrobial activity, compared to the
untreated controls. IL-8 activates neutrophils to increase their
numbers at the infected site. In the same experiment, IL-1 was
decreased significantly (over 300%). IL-1 increases inflammation
and decreases healing times. This test clearly showed that SRF and
the immunostimulators regulated the inflammatory process in dermal
tissues in a manner that would increase the body's ability to fight
infected wounds and increase the body's ability to healing quicker.
The same experiment was done with virally infected cells and the
SRF in combination with immunostimulators decreased viral plaque
formed by HSV-1 or 2 by 70% and HPV-18 decreased by 82%. Viral
plaques are a direct measure of viral numbers in infected cells.
The antiviral drug, Acyclovir also decreased viral HIV plaques by
60% and SRF with an immunostimulators and acyclovir, totally
eliminated the virus from the infected cells.
[0118] Percentage of Viral Plaque Reduction in Virally Infected
Cells in Humans
[0119] A total of 15 patients with vaginal HPV 16 or 18 were
treated with the SRF cream that also contained imiquimod. The
results clearly show that SRF and Imiquimod increased inflammatory
cytokines high enough needed to kill high numbers of the virus in
virally infected cells as measured by reduction in viral plaques.
Imiquimod normally reduced viral plaques in virally infected cells
by 25-40%. SRF reduces viral plaques in virally infected cells by
22-34%. Unexpectedly, the combination of SRF and Imiquimod produced
the best results, by reducing the viral plaques over 92%,
eliminating the virus from most of the infected cells. This tissue
culture data along with data from humans, confirms that this
combination worked.
Example 23 Lip Plumping Products the Addition of SRF Increases
Collagen Deposition and Added Better Moisturizing to the Lips
[0120] Alpha-Keto-glutarate is taken up by lip cells to increase
collagen deposition by fibroblasts. In combination with SRF,
Alpha-keto-glutarate produced the greatest results. This product
will plump up the lips and increase their thickness by increasing
collagen deposition. When you need a fast fix for thin lips, this
moisturizing gloss immediately stimulates your kisser with
cinnamon, peppermint and ginger to trigger plumping and flushing
with a hint of color and shine. Full, smooth & kissable soft.
Plumping agents used are cinnamon, capsaicin, caffeine, ginger,
menthol, niacin and vitamin A.
Example 24 Post Waxing or Shaving Product. Brazilian Chill
[0121] Post shaving gel with SRF produced a superior product. It
reduces the number of ingrown hair, razor bumps, and redness from
shaving, waxing, electrolysis or laser hair removal and is great
for women after shaving legs, bikini lines and underarms as well as
for men who see redness after shaving their faces. SRF reduces the
redness, erythema and pain. SRF reduced redness, pain erythema that
other products do not provide. SRF also increases healing of sore
irritated skin. Acetylsalicylic Acid--active ingredient that
unblocks pores and soothes skin.
Example 25 Cellulite
[0122] Cellulite is a skin condition found in 90% of women over 30
years of age. Skin adipocytes undergo hypertrophy and hyperplasia
thus cellulite is characterized by large stable adipocytes that are
limited to the lower body area. Many techniques have been tried,
but to date no real effective treatment exists. L arginine,
cinnamon oil, methysulfonylmethane have been tried with little
success. Stimulating Nitric oxide is effective to prevent and
reduce cellulite, reduce inflammation and increase
microcirculation. SRF was used in conjunction with Vitamin A
palmitate (retinol) acetyl dipeptide-1 cetyl ester, L arginine,
palmitoyl pentapeptide, sodium hyaluronate in the Genederm deliver
formula. In tests on 9 women with cellulite the SRF formula reduced
cellulite by 30% in a month as measured by photo imaging. In three
months, the cellulite was reduced by 56% as averaged over the 9
women treated.
[0123] The invention being thus described; it will be obvious that
the same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the invention
and all such modifications are intended to be included within.
* * * * *